Chapter 5 Enzymes
One. overview
Properties of enzymes Classification of enzymes
Two. Structure of the enzyme
Simple enzymes Binding enzymes Coenzymes and co-group monoenzymes Oligomerase multienzyme systems Active center Isoenzymes
Three. Catalytic mechanism of enzymes
Induced fit hypothesis Factors that accelerate the reaction speed of enzymes
Four. Enzymatic onset kinetics
Mie's equations Mi's constant and significance Of bi-bi reactions Factors influencing enzymatic reactions Activators inhibitors Competitive inhibition Non-competitive inhibition
Five. Regulation of enzymes
Allosteric regulation Covalent modification regulates eszyme progen activation
Section 1 Overview
1. Definitions
An enzyme is a biological catalyst and a protein that has a catalytic function.
Second, people's understanding of enzymes
In 1833, Payen and Persoz extracted from malt a heat-sensitive substance that hydrolyzes starch into soluble sugars called diastase, which means "separation." Therefore, later generations often add the ending of the word -ase to the name of the enzyme. Since they purified the cell-free enzyme preparation by ethanol precipitation and other methods, and discovered the catalytic properties and thermal instability of the enzyme, it is generally believed that they first discovered the enzyme.
Western studies of the phenomenon of fermentation in the 19th century led to further study of enzymes. Pasteur coined the term "enzymes", arguing that only living yeast cells could ferment. The term "ferment" is also often used in Japan. In 1878, the German Kuhne proposed the word "Enzyme", which means "in yeast". In 1896, the German Brothers Buchner ground yeast cells with quartz sand and obtained a cell-free filtrate that could catalyze fermentation, proving that fermentation is a chemical reaction and has nothing to do with cell viability. The discovery touches on the nature of enzymes, which some consider to be the beginning of enzymatic research.
In 1913, Michaelis and Menten used physical and chemical methods to propose the kinetic principle of enzymatic reactions, the Mi's theory, so that enzymology can be quantitatively studied. In 1926, American J.B. Sumner crystallized urease (the first enzyme crystallization) from knife beans and proposed that enzymes are proteins. Later, a variety of enzyme crystals were obtained to prove this idea, and Sumner won the 1947 Nobel Prize. Since then, a variety of enzymes have been discovered, crystallized, and structured, and sub-disciplines such as enzyme engineering have been produced.
After entering the 1980s, ribozyme, antibody enzymes, mimetic enzymes, etc. appeared one after another, and the traditional concept of enzymes was challenged. In 1982, Cech et al. discovered that tetrahyptode 26S rRNA precursors have self-splicing functions, and in 1986 demonstrated that its intron L-19 IVS has a variety of catalytic functions. Since then, a variety of RNAs with catalytic functions have been discovered, and the substrates have also expanded to DNA, sugars, and amino acid esters. Others have designed the synthesis of new ribosases in the lab. Peptide antibiotics such as bleomycin have even been found to have catalytic power. These new discoveries not only increase the study of the nature of enzymes, but also contribute to the exploration of issues such as the origin of life, making enzymatic research enter a new stage.
Third, the characteristics of enzymes
Enzymes are catalytic proteins produced by living organisms. 90% of proteins in cells are catalytically active. An enzyme is a biological catalyst that, like a general catalyst, only changes the reaction speed, does not change the chemical equilibrium, and does not change itself before and after the reaction. However, enzymes, as biological catalysts, have the following characteristics compared with general inorganic catalysts:
1. High catalytic efficiency The catalytic efficiency of enzymes is 106-1013 times higher than that of inorganic catalysts. For example, 1mol of equine liver catalase can catalyze the decomposition of 5×106 moles of hydrogen peroxide under certain conditions, and under the same conditions, 1mol of iron can only catalyze the decomposition of 6×10-4 moles of hydrogen peroxide. Therefore, the catalytic efficiency of this enzyme is 1010 times that of iron. That is, a reaction catalyzed with catalase in 1 second, the same amount of iron takes 300 years to complete.
2. Strong specificity General catalysts do not have strict requirements for substrates, can catalyze a variety of reactions, while enzymes only catalyze a reaction of a certain type of substance to generate specific products. Therefore, the types of enzymes are also diverse. Enzyme-catalyzed reactions are called enzymatic reactions, and the reactants of enzymatic reactions are called substrates. Enzymes only catalyze a specific reaction of a certain type of substrate to produce a certain product, which is called the specificity of the enzyme.
The specificity of various enzymes is different, including two categories of structural specificity and three-dimensional specificity, and structural specificity is divided into absolute specificity and relative specificity. Absolute specificity means that an enzyme catalyzes only one substrate to produce a definite product. Such as amino acids: tRNA ligase, only catalyze the linkage reaction of one amino acid to its receptor tRNA. Relative specificity refers to the reaction of enzymes that catalyze a class of substrates or chemical bonds. For example, alcohol dehydrogenase can catalyze the oxidation of many alcohols. There are also many enzymes with stereoscopic specificity, which have strict requirements for the configuration of the substrate. For example, lactate dehydrogenase can only catalyze L-lactate, not D-lactate reactions.
3. Mild reaction conditions Enzymatic reaction does not require high temperature and pressure and strong acid and alkali and other severe conditions, at room temperature and pressure can be completed.
4. The activity of enzymes is regulated by a variety of factors The catalytic capacity of inorganic catalysts is generally unchanged, while the activity of enzymes is affected by many factors. For example, the concentration of substrates and products, pH and the concentration of various hormones have a greater impact on enzyme activity. Changes in enzyme activity enable enzymes to adapt to the complex and varied environmental conditions and diverse physiological needs of the organism. Organisms regulate the metabolism of the body through allogeneic, enzymatic activation, reversible phosphorylation, etc.
5. Poor stability Enzymes are proteins that can only function under conditions of normal temperature, atmospheric pressure and near neutrality. High temperatures, high pressures, strong acids, strong bases, organic solvents, heavy metal salts, ultrasonic waves, vigorous agitation, and even the surface tension of the foam can inactivate enzymes. However, enzymes in nature are diverse, and some can work under extreme conditions. Some bacteria live under extreme conditions, such as superpyrogenes can live in an environment above 90 °C, the high limit is 110 °C; the maximum temperature of cold phages is -2 °C, which cannot grow above 10 °C; acid phages live below pH1, and the optimal pH of alkali phages is greater than 11; and the maximum temperature of phagocytophages can tolerate 1035 atmospheres. These phages have normal intracellular enzymes, but extracellular enzymes can tolerate extreme conditions. Some enzymes in organic solvents can catalyze reactions that cannot be accomplished in the aqueous phase.
Fourth, the naming and classification of enzymes
1. Naming
There are two ways to nomencate enzymes: custom nomenclature and system nomenclature. Customary naming is based on the substrate and reaction type of the enzyme, sometimes with the source of the enzyme. Customary naming is simple and commonly used, but lacks systematicness and is inaccurate. The 1961 International Enzymology Conference proposed a systematic nomenclature of enzymes. It is stipulated that the substrate of the enzyme and the type of reaction should be indicated, and the two substrates should be separated by a colon, and water can be omitted. The system name for ethanol dehydrogenase is: alcohol: NAD + oxidortase.
2. Classification
According to the type of catalytic reaction, the International Enzymology Committee divides enzymes into six categories. In these six categories, they are divided into a number of subcategories, and subclasses are divided into groups. Subclass classification criteria: redoxtase is the type of electron donor, transfer enzyme is the shape of the transfer group, hydrolases are the type of bonds that are hydrolyzed, lyases are the types of bonds that are lysed, isomerases are the types of isomeric action, and synthases are the types of bonds generated.
(1) Oxidortase catalyzes redox reactions, such as glucose oxidase, various dehydrogenases, etc. It is the largest class of enzymes that have been found, and its oxidation, capacity, and detoxification functions are second only to hydrolases in production. Cofactors are required and can be determined according to the change in the photoelectric properties of the cofactors at the time of reaction. According to the system name, it can be divided into 19 subcategories, and customarily can be divided into 4 subcategories:
²Dehydrogenase: the receptor is NAD or NADP, does not require aerobics.
² Oxidase: taking molecular oxygen as the receptor, the product can be water or H2O2, often requiring flavin auxiliary groups.
²Peroxygenase: H2O2 as the receptor, often with flavin and heme as auxiliary groups
²Oxygenase (oxygenase): Catalytic oxygen atoms incorporated into organic molecules, also known as hydroxylase. According to the number of oxygen atoms incorporated, it can be divided into single oxygenase and double oxygenase.
(2) Transfer enzymes catalyze the transfer reaction of functional groups, such as various transaminases and kinases to catalyze the transfer of amino and phosphate groups, respectively. Transferases, also called transferases, require coenzymes, but reactions are not easy to determine. According to the nature of transfer groups, it can be divided into 8 subcategories, the more important ones are:
² One carbon-based transferase: transfers one carbon unit, which is related to nucleic acid and protein methylation.
² Phosphate transferase: often referred to as kinase, mostly with ATP as a donor. A small number of proteases are also called kinases (such as incretin).
² Glycosidase: closely related to polysaccharide metabolism, such as glycogen phosphorylase.
(3) Hydrolases catalyze the hydrolytic reaction of substrates, such as proteases, lipases, etc. It plays a degrading role and is mostly located in extracellular or lysosomal. Some proteases are also called kinases. It can be divided into 11 subcategories of hydrolyzed ester bonds (such as restriction enzymes), glycosidic bonds (if gelase, lysozyme, etc.), peptide bonds, carbon and nitrogen bonds, etc.
(4) Lyases catalyze the reaction of removing a small molecule from the substrate leaving a double bond or its inverse reaction. Including aldolase, hydrase, decarboxylase and the like. There are 7 subcategories in total.
(5) Isomerases catalyze the mutual transformation between isomers. Including race enzymes, isomerases, metastases and the like. There are 6 subclasses in total.
(6) Synthases Catalysis is the synthesis of a substance by two substances, which must be coupled with ATP decomposition. Also called ligases, such as DNA ligases. There are 5 subclasses in total.
3. Number of enzymes
The International Enzymology Commission assigns a uniform number to each enzyme according to its category. The number of the enzyme consists of EC and 4 numbers separated by dots. EC represents the Enzymology Committee, the first number indicates the class of the enzyme, the second number indicates the subclass of the enzyme, the third number represents the group of enzymes, and the fourth number indicates the serial number of the enzyme in the group. For example, EC1.1.1.1 indicates that the enzyme is oxidoractase, the electron donor is an alcohol, the electron receptor is NAD+, and the serial number is 1, that is, ethanol dehydrogenase. The number of trypsin is EC3.4.4.4, and the four numbers indicate that its type is hydrolase; the hydrolyzed bond is a peptide bond; it is an endonuclease instead of an exonuclease; and the serial number is 4. Multifunctional enzymes can have multiple numbers.
Fifth, the vitality of enzymes
1. Definition refers to the ability of enzymes to catalyze certain chemical reactions.
2. Units Under specific conditions, the amount of enzyme required to transform 1 micromolal substrate in 1 min is one viability unit (U). The temperature is specified at 25 degrees, and other conditions take the most suitable conditions for the reaction.
Specific live: The enzyme activity per milligram of enzyme protein. The unit is u/mg. The higher the specific activity, the more pure the enzyme.
Number of transformations: The number of substrate molecules (TN) that can be catalyzed per molecule or per enzyme active center per unit time. This corresponds to the speed constant kp of the enzymatic reaction. Also known as the catalytic constant (Kcat). 1/kp is called the catalytic cycle. Carbonic anhydrase is one of the highest known conversion enzymes, up to 36×106 per minute, with a catalytic cycle of 1.7 microseconds.
3. Determination The method of determining the initial velocity of the enzymatic reaction is generally used to determine the activity, because there are fewer interfering factors at this time and the speed remains constant. The unit of reaction velocity is concentration/unit time, which can be expressed as the amount of substrate reduction or product increase. Because the product concentration changes greatly from scratch, and the substrate is often excessive, its change is not easy to measure accurately, so the product is mostly used to determine.
Section 2 Structure of enzymes
First, the chemical composition of enzyme molecules
The essence of an enzyme is a protein. Enzymes, like other proteins, are made up of amino acids and have a primary, secondary, tertiary, and quadranic structure. Enzymes can also be denatured by certain physical and chemical factors and lose vitality. The enzyme has a large molecular weight and colloidal properties and cannot be dialyzed. Enzymes can also be hydrolyzed by proteases.
1. Cofactors
Some enzymes are completely composed of proteins and belong to simple proteins, such as ureases, proteases, etc.; some enzymes contain non-protein components in addition to proteins, which belong to binding proteins. The non-protein components are called cofactors, the protein part becomes the enzyme protein, and the complex is called the whole enzyme. Cofactors generally play a role in carrying and transferring electrons or functional groups, wherein the tight binding with enzyme proteins with covalent bonds is called auxiliary groups, and the loose binding with non-covalent bonds is called coenzymes.
In the catalytic process, the auxiliary group is not separated from the enzyme protein, and only acts as an intra-enzyme carrier, such as fad, FMN co-radicals in flavin protein enzyme molecules carry hydrogen, and biotin co-groups of carboxylases carry carboxyl groups and so on. Coenzymes often act as interenzymatic carriers, linking two enzymatic reactions, such as NAD+ being reduced to NADH in one reaction and oxidized back to NAD+ in another. It behaves like substrates in reactions, sometimes referred to as auxiliary substrates.
More than 30% of enzymes require metal elements as cofactors. The metal ions of some enzymes bind tightly to enzyme proteins and are not easy to separate, called metal enzymes; the metal ions of some enzymes are loosely bound, called metal activation enzymes. The cofactors of metal enzymes are generally transition metals, such as iron, zinc, copper, manganese, etc.; the cofactors of metal activation enzymes are generally alkali metals or alkaline earth metals, such as potassium, calcium, magnesium and the like.
2. Monomeric enzymes, oligomerases and multi-enzyme systems
Enzymes composed of a peptide chain are called monomer enzymes, and enzymes formed by the binding of multiple peptide chains with non-covalent bonds are called oligoenzymes, which belong to oligopolyptins. Sometimes in the organism, some functionally related enzymes are organized to form a multi-enzyme system, which in turn catalyzes the relevant reactions. Constituting a multi-enzyme system is metabolically necessary to reduce diffusion restrictions on substrates and products and increase the speed and efficiency of the total reaction.
Sometimes there are multiple enzyme activities on a peptide chain called multi-enzyme fusions. For example, the debranching enzyme in glycogen decomposition has starch-1,6-glucosidase and 4-α-D-dextran transferase activity on a peptide chain; camphorin from camphorin from camphor seed consists of a peptide chain and has three activities: (1) RNA N-glycosidase activity, which hydrolyzes the glycosid bond of adenylate at position 4324 in rat 28S rRNA, releasing an adenine; (2) endonuclease activity dependent on the superhel DNA configuration, Specifically despin and cleave superhelix cyclic DNA to form notched ring-like and linear DNA; (3) superoxide dismutase activity. The AROM multi-enzyme fusion from Streptomyces redis is a dimer containing five enzyme activities per peptide chain, which can catalyze the second to sixth steps of the shiroxychloric acid pathway, and the catalytic efficiency is greatly improved due to the transmission channel of the intermediate product.
Second, the active center of the enzyme
1. Definitions
Enzymes are macromolecules, and their molecular weight is generally more than 10,000, which is composed of hundreds of amino acids. The substrate of the enzyme is generally very small, so only a small part of the enzyme molecule is in direct contact with the substrate and plays a catalytic role. Although the substrate of some enzymes is large, it is only a small area in contact with the enzyme. Therefore, it is believed that there is an active center in the enzyme molecule, which is a small part of the enzyme molecule, which is the place in the enzyme molecule that binds to the substrate and catalyzes the reaction. The center of activity is composed of a few amino acid residues in the enzyme molecule, which may be very far apart in the primary structure, or even located on different peptide chains, due to the coiled folding of the peptide chain and close to each other, constituting a specific active structure. Therefore, the center of activity is not a point or surface, but a small area of space.
The amino acids in the active center can be divided into substrate binding sites and catalytic sites according to function. The former is responsible for identifying and binding to a particular substrate. They determine the substrate specificity of the enzyme. The catalytic site is catalytic, where the sensitive bonds of the substrate are cut off or new bonds are formed and a product is generated. The difference between the two is not absolute, and some groups have both substrate binding and catalytic functions.
Koshland divides the residues in enzyme molecules into four categories: contact subunits are responsible for substrate binding and catalysis, auxiliary subunits play an assisting role, structural subunits maintain the conformation of enzymes, and substitution of non-contributing subunits has no effect on activity, but has an effect on enzyme immunity, transport, regulation and lifespan. The first two constitute the center of activity, and the first three are called the necessary groups of enzymes.
The parts outside the active center are not useless, they are able to maintain the spatial structure of the enzyme, so that the active center remains intact. After the enzyme binds to the substrate, the conformation of the entire enzyme molecule changes, and this twisted tension makes the chemical bond of the substrate easily broken. This change also relies on the synergy of inactive centers.
In general, monomeric enzymes have only one center of activity, but some multifunctional enzymes with multiple functions have multiple centers of activity. For example, E. coli DNA polymerase I is a 109kd peptide chain that has both polymerase activity and exonuclease activity.
3. Formation process
Some enzymes are not yet catalytically active when they are just synthesized or secreted in cells, and the precursors of these inactive enzymes are called enzymaticogens. Proenzymes are activated to convert into active enzymes. Activation of the enzyme is a mechanism to control the activity of the enzyme by changing the covalent structure of the enzyme molecule, through the shearing of the peptide chain, changing the conformation of the protein, thereby forming or exposing the active center of the enzyme, so that the enzyme is activated as an active enzyme if necessary, playing its function.
4. Methods for studying active centers
The structural characteristics of enzyme substrates and competitive inhibitors help to study the structure of the active centers of enzymes. Kinetic characteristics such as the optimal pH and velocity constant of the enzyme also provide some information.
Chemical modifications have played an important role in the study of active centers. Because the group reactivity of the active center is often different from other groups, some reagents may specifically react with a residue in the active center, rather than with the residue outside the active center. For example, DFP (diisopropylfluorophosphate) can react with serine in the active center. TPCK (N-p-toluenesulfonylphenylpropanyl chloromethyl ketone) is more specific and can only bind to His-57 at the epicenterase activity center, called affinity marker. It is a substrate analogue, an alkylating agent. TLCK (lysine derivative) acts on his-46 of the pancreatic enzyme. Some reagents are not specific, can be used differential labeling: first with substrate analogues to protect the active center, add modifiers, react with groups outside the active center, and then remove the inhibitor, and then add radioactive labeled reagents, at this time the reagent can only react with the group of the active center, determine the position of the radioactivity, you can find the active center.
Ultraviolet, fluorescence, garden dichroic spectroscopy and other methods can also be used with the study of active centers. When the enzyme binds to the substrate, the chromophore located at the substrate binding site will inevitably undergo some kind of change, resulting in a change in its spectrum. These chromophores can be carried by the enzyme itself or introduced artificially. This method can be used to determine the composition of the active center, and it can also be used to study the catalyzed reaction process. The most direct and accurate method is X-ray diffraction.
3. Isoenzymes
Isoenzymes are different enzyme molecules that catalyze the same reaction by the same organism. Isoenzymes have the same catalytic effect, but their functional significance is different. Enzymes that have the same function in different organisms are not isoenzymes. Isoenzymes have the same or similar centers of activity, but their physicochemical and immunological properties are different. Cell localization, specificity, activity, and regulation of isoenzymes can vary. Each isoenzyme has its own unique functional significance. For example, lactate dehydrogenase (LDH) is a tetramer composed of 4 subunits. There are two subunits, A(M) and B(H), and there are 5 isoenzymes: LDH1 (H4), LDH2 (MH3), LDH3 (M2H2), LDH4 (M3H), LDH5 (M4). The two subunits M and H are encoded by different genes, and the synthesis speed in different cells is different, so the proportion of 5 isoenzymes in different tissues and organs is different, and different isoenzyme profiles will be obtained after electrophoresis separation. There is more LDH1 and LDH2 in the human heart muscle, and more LDH5 in skeletal muscle. M subunit has a higher Km of pyruvate and is not inhibited by substrates, so muscles can produce a large amount of lactic acid; H subunit Km is small, and is inhibited by substrates, which saturates quickly with the increase of substrates, so the heart produces very little lactic acid, and this enzyme is mainly used for the oxidation of lactic acid. Clinically, the analysis of the patient's serum LDH isoenzyme profile helps to diagnose the site of lesion occurrence. For example, serum LDH1 is elevated in myocardial damage and LDH3 is elevated in lung damage.
Section 3 Catalytic Mechanism of Enzymes
First, the binding of enzymes to substrates
The forces of binding of enzymes to substrates are mainly hydrogen bonds, salt bonds and van der Waals forces. Salt bonds are the electrostatic attraction between charged groups, and the action between hydrophobic groups is also called hydrophobic bonds.
The combination of enzymes and substrates is specific, and people used to use locks and keys to compare the relationship between enzymes and substrates. This "key lock theory" is incomplete. For example, enzymes can bind to both substrates and products to catalyze their inverse reactions. Therefore, the "induced fit theory" was proposed, that when the enzyme is close to the substrate, the enzyme protein is induced by the substrate molecule, and its conformation changes, becoming conducive to binding and catalysis with the substrate.
Second, the enzyme speeds up the reaction speed of the factor
Enzymes speed up the reaction mainly by reducing the activation energy of the reaction, that is, the energy required for the substrate molecules to reach the activation state. For example, urease can reduce the activation energy of the urea hydrolysis reaction from 136kj/mol to 46kj/mol, increasing the reaction speed by 1014 times. The catalytic mechanism of enzymes mainly includes the following points:
1. Proximity orientation In response to a bimolecular reaction, the enzyme can make the two substrates bind to each other in the active center and have a certain orientation. This reacts more easily than random collisions in solution. For different reactions, after changing from an intermolecular reaction to an intramolecular reaction, the reaction speed can be accelerated by 100 to 1011 times.
2. Substrate deformation When an enzyme binds to a substrate, not only the conformation of the enzyme changes, but also the conformation of the substrate. This change brings the substrate closer to the transition state and therefore reduces the activation energy.
3. Acid-base catalysis and covalent catalysis The side chain groups of some residues in the enzyme activity center can play the role of acid-base catalysis or covalent catalysis. Acid-base catalysis can be divided into two kinds: general acid-base catalysis and special acid-base catalysis, special acid-base catalysis refers to the catalytic effect of H+ and OH-; general acid-base catalysis also includes the catalysis of other weak acids and weak bases. Enzymatic reactions generally occur in near-neutral conditions, and the concentrations of H+ and OH- are very low, so enzymatic reactions are mainly generally acid-base catalyzed. Some dissociable groups in enzyme molecules such as imidazolyl, carboxyl, amino, and mercapto often play a general acid-base catalytic role, of which imidazole is the most active and effective.
Some enzymes have acid-base cocatalytic mechanisms and proton transfer pathways. The rotation reaction of tetramethyl glucose in benzene is slow if it is catalyzed by pyridine (base) or phenol (acid) alone; if the two are mixed catalyzed, the speed is accelerated, that is, acid-base cocatalysis. If the acid and base are concentrated in one molecule, that is, the synthesis of α-hydroxypyridine, its catalytic speed is accelerated by another 7,000 times. This is because two catalytic groups concentrated in one molecule facilitate the transport of protons. In enzyme-substrate complexes, hydrogen bonds and conjugate structures often form proton transport pathways, thereby greatly improving catalytic efficiency.
Covalent catalysis can be divided into electrophilic catalysis and nucleophilic catalysis. Serine proteases, papain containing thiol groups, and pyruvate decarboxylases with thiamine as a coenzyme all have nucleophilic catalytic effects. Hydroxyl, mercapto, and imidazolyl all have nucleophilic catalysis. Metal ions and tyrosine hydroxyl groups, -NH3+ are electrophilic groups. Covalent catalysis often forms covalent intermediates with high reactivity, turning one-step reactions into two- or multi-step reactions, bypassing higher barriers and making reactions progress quickly. For example, trypsin forms an acyl-enzyme covalent intermediate through the hydroxyl groups of the serine side chain, reducing the activation energy.
4. The role of the microenvironment The active center of some enzymes is a hydrophobic microenvironment, which has a low dielectric constant, which is conducive to the interaction between the charges, and also conducive to the formation and stability of intermediates. For example, the pK of the lysine side chain amino group is about 9, and the pK of the lysine side chain in the center of acetylactic acid decarboxylase activity is only about 6.
All of the above points can speed up the reaction, but each enzyme is different and can have several of these factors at the same time.
Section 4 Kinetics of Enzymatic Reactions
The kinetics of enzymatic reactions is the science of studying the speed of enzymatic reactions and the various factors that affect their speed. Kinetic studies can not only provide experimental evidence for the study of the mechanism of enzymes, but also guide the application of enzymes in production, and maximize the catalytic effect of enzymes.
1. Mr. U.S.
1. Derivation of Mi's equations
The Michaelis and Menton theory was established in 1913, arguing that the reaction is divided into two steps, mr. enzyme -substrate complex (intermediate product), and then decomposed to form a product, releasing free enzymes. After being supplemented and developed by Briggs and Haldane, the current Michaelis equation was obtained.
S-E-SE-P-E
For the above reaction, there are first three assumptions: first, the substrate is overdose, that is, [S]"[E]. Second, in the early stage of the reaction, the product concentration is extremely small, ignoring the inverse reaction that is, k-2 = 0; third, the steady-state hypothesis, that is, as the reaction progresses, the formation rate of the complex gradually decreases, the decomposition accelerates, and equilibrium is reached at a certain time, and the concentration of the complex is constant, which is called "steady state". After the system reaches a steady state, the consumption of substrates and the rate of product generation are constant and equal. After the assay, the enzyme is added to the system and can reach the steady state within a few milliseconds, so the initial velocity we measure is usually the steady-state velocity. The system remains steady-state until the product accumulates more, so the reaction rate
v=k2[ES]。 According to the steady-state hypothesis , there is k1[E][S] = (k-1 + k2) [ES], i.e. [ES] = k1[E] [S] / (k-1 + k2). Definition (k-1 +k2)/k1=Km, because [E]= [E]0-[ES], so [ES]= [E]0[S]/(Km+[S]). Substitute the velocity equation to get v= k2[E]0[S]/(Km+[S]). Because the speed is greatest when [ES]=[E]0, Vm=k2[E]0. Substitute to get the following Mihn's equations:
v=Vm×[S]/(Km+[S])
2. The meaning of the Michaelis constant
The physical significance of the Mitchell constant is the concentration of the substrate at which the reaction velocity reaches half the maximum reaction velocity. Its enzymatic significance lies in the fact that it is a characteristic constant of the enzyme, which is only related to the nature of the enzyme and has nothing to do with the enzyme concentration. Different enzymes have different Km, and the same enzyme is different for different substrates. At k2 polar hours 1/Km can be approximated to indicate the affinity of enzymes and substrates, the greater the 1/Km, the greater the affinity. Among the various substrates of an enzyme, the smallest substrate of Km is called the natural substrate of the enzyme.
3. Determination of the Michael's constant
Vm can be obtained from the v-[s] plot of the enzyme, and then read out [s] from 1/2Vm, that is, Km. But in fact, it can only be infinitely close to Vm, but it cannot be reached. In order to obtain an accurate Mihn's constant, the Mi's equation can be deformed so that it is equivalent to a linear equation, and the accurate Mi's constant can be obtained by plotting.
Double reciprocal diagramming rewrites the equation as
1/v=Km/Vm×1/[S]+1/Vm
During the experiment, the initial velocity was determined at different substrate concentrations, and the plot was 1/v vs. 1/[S], and the straight line was extrapolated to intersect the horizontal axis, and the horizontal axis was intercepted as -1/Km, and the vertical axis was intercepted as 1/Vm. This method is called the Lineweaver-Burk graphing method, which is the most widely used, but the experimental points are often concentrated at the left end, and the drawing is not easy to be accurate.
The Eadie-Hofstee method rewrites the equation as
v=-Km×v/[S]+Vm
Plot in v vs v/[S] with a straight slope of -Km.
4. Other kinetic parameters
Kcat/Km is called the specificity constant of the enzyme, which is not affected by non-productive binding and intermediate product accumulation, and can indicate the specificity of the enzyme to several competing substrates. The concentration of substrates for many reactions under physiological conditions is very low. At very low substrate concentrations, v= (Kcat/Km)[E][S], i.e., Kcat/Km is the apparent secondary velocity constant. Because Kcat /Km = k3k1 / (k2 + k3), it is less than k1, that is, less than the velocity constant of enzyme and substrate complex formation. It is not a true microscopic velocity constant, and it is only true when the speed-limiting step of the reaction is a collision of enzymes with substrates. The diffusion limit determines that the upper limit of the velocity constant is 108-109mol-1s-1, and carbonic anhydrase, propionose phosphate isomerase, acetylcholinesterase, etc. are close to this limit, indicating that their evolution has been perfected.
Reaction stage: for the reaction of xA + yB = p, the velocity v = k [A]a[B] b, for substrate A is grade a, for substrate B is b grade, the whole reaction series is a + b grade. The number of reaction molecules refers to the minimum number of molecules participating in the slowest step of the reaction. It refers to the reaction mechanism and must be an integer; the reaction series is measured experimentally and can be a decimal number. According to the Mihnov equation, when the substrate concentration is much greater than the Mi's constant, v=Vm is a zero-level reaction; conversely, v=(Vm/Km)[s], which is a first-order reaction. The middle part is a mixed-grade reaction.
Second, the mechanism of multi-substrate reaction
Many enzyme-catalyzed reactions are more complex, containing more than one substrate, and their reactions are divided into several categories according to the number of molecules, single molecules called uni, bi molecules called bi, three molecules are ter, and four molecules are quad. More common is a two-substrate double-product reaction called a bi-bi reaction:
A+B→P+Q
It is currently thought that most two-substrate reactions may have three reaction mechanisms:
1. Sequential reaction mechanism
Reactions requiring DEHYDROGENases for NAD+ or NADP+ fall into this category. Coenzyme as substrate A first with the enzyme to generate EA, and then with substrate B to generate ternary complex EAB, dehydrogenation to form product P, and finally release reduced coenzyme NADH or NADPH.
2. Stochastic mechanism
The addition of substrates and the release of products are random and have no fixed order. Such as glycogen phosphorylation reaction.
3. Ping-Pong mechanism
Aminotransferase is a typical ping-pong mechanism, the enzyme first interacts with substrate A (amino acid), produces an intermediate product EA, the amino group in the substrate is transferred to coenzyme, so that pyridoxal phosphate in the coenzyme becomes pyridoxine phosphate, that is, EA is converted into FP, and then the product P (α-ketoic acid) is released, to obtain enzyme F, and then interacts with substrate B (another ketoic acid), releasing product Q (corresponding amino acid) and enzyme E. The reaction of acetyl-CoA, ATP and HCO3-substrates to generate propionyl-CoA is also part of the ping-pong mechanism.
Third, the factors affecting the reaction speed
(A) the influence of pH Most of the activity of enzymes is affected by pH, and the activity is the highest at a certain pH value, which is called the most suitable pH. The optimal pH of general enzymes is 6-8, and a few enzymes require acidic or alkaline conditions. For example, pepsin is optimal pH at 1.5, while hepatic arginase is at 9.7.
pH affects the conformation of enzymes, as well as the dissociation status of the catalytic-related groups and the dissociation state of the substrate molecules. Optimal pH sometimes varies depending on substrate type, concentration, and buffer solution composition, and is not completely unchanged.
The pH-enzyme viability curve of most enzymes is a bell curve, but there are also a few enzymes that are only half the shape of the bell, or even straight lines. For example, the change in charge of the papain substrate has no effect on catalysis, and there is a straight line between pH4-10.
(II) The influence of temperature The curve of enzyme activity with temperature change is a bell curve, which has a highest point, that is, the most suitable temperature. The optimal temperature of enzymes in warm-blooded animals is 35-40 degrees, and plant enzymes are at 40-50 degrees. This is the result of the comprehensive balance of accelerated chemical reactions (1-2 times faster reaction speed per 10 °C per temperature increase) and enzyme inactivation at elevated temperatures. Generally, enzymes are denatured at more than 60 degrees, and a few enzymes can withstand high temperatures, such as bovine pancreatic ribonuclease heated to 100 degrees and still not inactivated. Dried enzymes tolerate high temperatures, while liquid enzymes are inactivated quickly.
The optimal temperature is also not a fixed value, it is affected by the reaction time, the enzyme can tolerate higher temperatures for a short period of time, and the optimal temperature is reduced when the time is extended.
The activation energy of heat inactivation is generally 50-100Kcal/mol, which is 10 times higher than the activation energy of the general reaction, and is stable below 30 °C.
(C) the effect of activators All substances that can improve the activity of enzymes are called activators. Most activators are ionic or simple organic compounds. According to the size of the molecule, it can be divided into three categories:
1. Inorganic ions can be divided into three types: metal ions, hydrogen ions and anions. The metal ions that act as activators are potassium, sodium, calcium, magnesium, zinc, iron, etc., and the atomic number is between 11-55, of which magnesium is an activator of a variety of kinases and synthases. The activation effect of anions is generally not obvious, and the more prominent is that the α amylase in animal saliva is activated by chloride ions, and the activation effect of bromine is slightly weaker.
The action of the activator is selective and may have an inhibitory effect on another enzyme. Some ions also have antagonistic effects, such as sodium inhibiting potassium activation, calcium inhibiting magnesium. Some metal ions can be substituted for each other, such as the magnesium ions of kinase can be replaced with manganese. The concentration of the activator also has an effect, and too high a concentration may have an inhibitory effect. For example, for NADP+ synthase, the magnesium ion concentration is activated at 5-10×10-3 M, and the enzyme activity decreases at 30×10-3 M.
2. Medium-sized organic molecules Certain reducing agents such as cysteine, reduced glutathione, cyanide, etc., can activate certain enzymes, open the disulfide bonds in the molecule, and improve enzyme activity, such as papain, D-glyceraldehyde-3-phosphate dehydrogenase, etc. The other is EDTA, which chelates metals and relieves heavy metals from inhibiting enzymes.
3. Protein refers to enzymes that can act on certain inactive enzymes.
(4) The role of inhibitors
The effect of reducing enzyme viability but not causing enzyme protein denaturation is called inhibition. Substances that can cause inhibition are called inhibitors of enzymes. The inhibitor reacts with certain essential groups on the enzyme molecule, causing a decrease in enzyme activity or even loss, but does not denature the enzyme. Studying inhibition contributes to the understanding of enzyme mechanisms, biological metabolic pathways, and drug mechanisms of action. Inhibition can be divided into two categories according to reversibility: reversible inhibition and irreversible inhibition.
1. Irreversible inhibition (irreversible inhibition) Such inhibitors usually bind to enzymes with covalent bonds and cannot be removed by dialysis, ultrafiltration and other methods. According to the selectivity of inhibitors, they can be divided into specific and non-specific irreversible inhibitors. The former can only react with groups at the active site, while the latter can react with a variety of groups. If the affinity for the active site group is three orders of magnitude greater than that of other groups, it is a specific inhibitor. Sometimes depending on the object and conditions of action, some non-specific inhibitors will be transformed, resulting in a specific inhibitory effect. Commonly used methods to judge specificity are: there is a substrate protection phenomenon, there is a stoichiometry relationship, and the enzyme is completely inactivated after action, and does not react with the inactivated enzyme.
Common irreversible inhibitors are:
² Organophosphorus compounds can be firmly bound to hydroxyl groups on serine directly related to enzyme activity, thereby inhibiting certain proteases and esterases, and are specific inhibitors. Such compounds strongly inhibit cholinesterase, causing acetylcholine to accumulate, causing a series of symptoms of neurotoxicity, also known as nerve agents. DFP and organophosphorus insecticides used in World War II fall into this category. When there is a large amount of substrate, the substrate first binds to the active part of the enzyme, and the inhibition effect is weakened, which is called substrate protection. Although organophosphorus does not dissolve after binding to the enzyme, sometimes the phosphate root on the enzyme can be removed by oxime (containing -CH=NOH group) or hydroxyxime acid, so that the enzyme can be restored. Clinically used antiphosphordaine (PAM) is one such compound.
² Organic arsenic, mercury compounds interact with thiol groups, inhibition of thiol-containing enzymes. For example, p-chloromercurybenzoic acid, may be relieved of excess thiol compounds such as cysteine or reduced glutathione. Arsenides disrupt lipoic acid coenzymes, thereby inhibiting the pyruvate oxidase system. Lewis poison gas (CHCl= CHAsCl2) inhibits almost all thiolase enzymes. The toxicity of arsenides can not be relieved with monomerthic compounds, and can be relieved by excessive amounts of bisthyl compounds, such as dimercaptyl propanol and the like. It is an important clinical antidote to arsenide and heavy metal poisoning.
²Cyanide binds to Fe2+ in iron-containing porphyrinases such as cytochrome oxidase, inactivating the enzyme and inhibiting cellular respiration.
² Heavy metals Salts such as silver, copper, lead, mercury and other salts can inactivate most enzymes and can be relieved by chelating agents such as EDTA. It may be a reaction with a thiol group in an enzyme molecule, or a replacement of metal ions in an enzyme.
² Alkylating agents are mainly halogen-containing compounds, such as iodoacetic acid, iodoacetamide, haloacetylbenzene, etc., is a non-specific inhibitor that can alkyl alkylate thiol and inactivate enzymes. Commonly used to identify thiol groups in enzymes.
²Suicide substrates exist in an incubation state, are activated after binding to the active centers of certain enzymes, producing an inhibitory effect. Such inhibitors are highly specific and only shift when encountering target enzymes. Also known as Kcat-type specialized irreversible inhibitors. Another class of specific irreversible inhibitors is called Ks type, which is a substrate analogue, such as TPK And the like.
2. Reversible inhibition The binding with the enzyme is reversible, and the inhibitor can be removed by dialysis to restore the enzyme activity. Depending on the relationship between inhibitors and substrates, reversible inhibition can be divided into three types:
(1) Competitive inhibition The structure of the inhibitor is similar to the substrate, and the enzyme forms a reversible EI complex but cannot be decomposed into product P. The inhibitor competes with the substrate for the center of activity, thus preventing the substrate from binding to the enzyme. This inhibition can be weakened by increasing the substrate concentration. The most typical example is the inhibition of succinic acid dehydrogenase by malonic acid. Competitive inhibitors increase Km, Km'=Km× (1+I/Ki), and VMs remain constant.
Competitive inhibition is most common, and sulfonamides are competitive inhibitors such as p-aminobenzenesulfa. It is similar to p-aminobenzoic acid and inhibits bacterial dihydrofolate synthase, thereby inhibiting bacterial growth and reproduction. The human body can use folic acid in food, while bacteria cannot use foreign folic acid, so they are sensitive to such drugs. The antibacterial synergist TMP enhances the efficacy of sulfonamide because its structure is similar to dihydrofolate, which inhibits bacterial dihydrofolate reductase, but rarely inhibits human dihydrofolate reductase. It is used in conjunction with sulfa to double hinder the synthesis of tetrahydrofolate by bacteria, seriously affecting the nucleic acid and protein synthesis of bacteria.
Certain alkaloids in plants such as phytostens are competitive inhibitors of cholinesterase and contain quaternary ammonium groups, similar to acetylcholine, which inhibit cholinesterase activity.
(2) Non-competitive inhibitory enzymes can bind to substrates and inhibitors at the same time, and there is no competition between the two. But the intermediates formed, ESI, cannot be broken down into products, so enzyme activity is reduced. Non-competitive inhibitors bind to groups other than the center of enzyme activity, mostly to thiol groups, destroying the conformation of enzymes, such as some compounds containing metal ions (copper, mercury, silver, etc.). Leucine is a non-competitive inhibitor of arginase, and inhibition caused by EDTA complexed metals is also non-competitive inhibition, such as inhibition of hexokinase that requires magnesium ions. Non-competitive suppression keeps Km constant and VMs smaller.
(3) Anti-competitive inhibition Enzymes can only bind to inhibitors after binding to substrates, and complexes cannot generate products. Anti-competitive inhibitors make both Km and Vm smaller.
Section 5 Regulation of Enzymes
Organisms control the rate of metabolism by regulating the function of enzymes. There are two types of regulatory mechanisms for enzymes, one is the regulation of the number of enzymes, and the other is the regulation of enzyme activity. The former controls the amount of enzyme by controlling the synthesis and degradation rate of enzymes, and the effect is slow and long-lasting, called coarse regulation; the latter changes the activity of enzymes, and the effect is fast and short, called fine tuning.
First, the regulation of enzyme activity
(1) Allosteric regulation
Some enzymes are induced by specific allosteric effectors to change their structure, so that the catalytic activity changes, called allosteric enzymes (allosteric enzymes). The effector that increases the activity of the enzyme is called a positive regulator, and vice versa is called a negative regulator. Alloenzymes are oligomerases, and in addition to the active centers in the molecule there are allosteric centers (regulatory centers). Both centers can be in the same subunit or in different subunits. Subunits with active centers are called catalytic subunits, and subunits with isomeric centers are called regulatory subunits. The allosteric effect can also be extended to non-enzymatic proteins, such as hemoglobin in the process of binding to oxygen.
The v-[S] curve of most allosterases is S-shaped, unlike Michizanase. This curve indicates that after the enzyme binds to a molecule of a molecular substrate (or effector), its conformation changes, favoring the binding of subsequent molecules, called positive synergistic effects. This phenomenon is conducive to the regulation of the reaction rate, and a slight increase in the concentration of the substrate when the maximum reaction speed is not reached will greatly increase the reaction rate. Therefore, the positive synergistic effect makes the enzyme extremely sensitive to changes in substrate concentration.
Another class of conformal enzymes has a negative synergistic effect, and its kinetic curve is similar to a hyperbolic curve, and the reaction rate changes quickly at low substrate concentrations, but slowly changes when it continues. Therefore, the negative synergistic effect makes the enzyme insensitive to changes in substrate concentration.
3. Judgment
Some enzymes without allosteric effects can produce similar curves, so the graphing method cannot be used as a basis for judging isomeric enzymes. Three enzymes can be quantitatively distinguished using rs (saturation ratio) ([S]90%V/[S]10%V): Rs equal to 81 is Mi's enzyme, greater than 81 has a positive synergistic effect, and vice versa is negative synergy. More commonly used is the Hill coefficient method, which plots log[S] in log(v/(Vm-v)), the maximum slope of the curve h is called the Hill coefficient, the Mihnase is equal to 1, the positive synergy enzyme is greater than 1, and the negative synergy is less than 1.
4. Machine
Homogeneous model (M. W.C.): It is believed that the conformation of all atoms in the enzyme molecule is the same, without heterozygous states. There is a balance between a low-activity tight (T) and a highly active relaxed form (R), where the effector moves the equilibrium, thereby altering the activity of the enzyme. This model is not suitable for enzymes with negative synergy.
Order variable model (K.N.F.): It is believed that individual subunits can be heterozygous, and that allosterism is due to the induction of ligands, rather than because of the movement of equilibrium. Synergy depends on the effect of subunits bound to ligands on vacant subunits. This model works for both enzymes.
5. Examples
(1) Aspartate transcarbamoylase (ATCase):
This is the first enzyme in the pyrimidine synthesis pathway, which is inhibited by the feedback of CTP and can be activated by ATP. Asp and carbamoyl phosphoric acid have positive homotropic effects, and CTP has heterotropic effects, which can increase the S-shape of the enzyme, that is, the Rs value decreases, and there is a positive synergy between TPPs, n=3. ATP increases Rs and becomes hyperbolic when saturation is reached. Both ATP and CTP only alter the affinity of the enzyme, not the Vm. Succinic acid is an analogue of aspartic acid, which is a competitive inhibitor at high aspartic acid concentrations, and can mimic the positive regulatory allosteric effect of aspartic acid as an activator when aspartic acid is insufficient.
This enzyme has a total of 12 subunits, of which 6 are catalytic and 6 are regulated subunits. The molecular structure is 2 C3 sandwiched between 3 R2, and the active center is located in the middle of two catalytic subunits. The allosteric center is located at the distal end of the regulatory subunit, which affects the activity of the catalytic subunit through allosterism.
(2) Phosphoglycerol dehydrogenase GDP
There are four subunits, Km1 and Km2 are smaller and easy to bind to NAD+, that is, react faster at low substrate concentrations, while Km3 is increased by 100 times, making it difficult to react with NAD+. This is caused by conformational changes. In organisms, fermentation can be guaranteed when NAD+ is insufficient, and other reactions are supplied when NAD+ is passed, avoiding acidosis.
(2) Covalent adjustment
This regulation is a transition between the active and inactive forms through enzymatic covalent modifications. The most typical example is glycogen phosphorylase in animal tissues, which catalyzes glycogen decomposition to produce glucose-1-phosphate. This enzyme comes in two forms: highly active phosphorylase a and lowly active phosphorylase b. The former is an oligomerase of four subunits, each containing a phosphorylated serine residue. These phosphate groups are essential for activity and can be water-removed under the action of phosphorylase phosphatase and become two lowly active hemimolecules: phosphorylase b. Phosphorylase b can accept the phosphate group of ATP to become phosphorylase a under the catalysis of phosphorylase kinase.
Covalent regulatory enzymes can amplify chemical signals. A molecule of phosphorylase kinase can catalyze thousands of phosphorylase b in a short period of time, and each phosphorylase a can catalyze the production of thousands of glucose-1-phosphates, thus constituting a two-step cascade of amplification. This is actually part of a longer cascade of amplifications in which epinephrine causes glycogen to break down dramatically.
Another class of covalent regulatory enzymes is E. coli glutamine synthase, etc., which are covalently modified by the adenosyl group transferred from ATP, or enzymatically de-adenosyl groups and regulate activity. In addition, the activation of the enzyme is also a covalent regulation.
(c) proenzyme (proenzyme; zymogen) activates
The protease secreted by the digestive tract is often secreted in the form of inactive progenases, which are activated when they reach their destination. This avoids hydrolysis of the digestive glands.
Pancreatin is originally cut by trypsin to produce π-pancreatin, π-xymotrypsin with high but unstable activity, and autophase cleavage produces a less active but stable α- pancreatin. After the activation of the original enzyme, the conformation changes, forming a hydrophobic pocket, that is, an active enzyme.
A complete center of activity has been formed in the protopepsinogen, but there is an alkaline sequence in the progenase that forms a salt bridge with the center of activity, blocking the center of activity. At pH below p5, the enzyme progen can be activated automatically, losing 44 fragments of the precursor of the residue. Activated enzymes can also reactivate other proenzymes.
Trypsinogen can be activated by incretin, followed by preypsinogen, propsinogen, prostase elastase, and protocarboxypeptase. So trypsin is a co-activator of the original trypsin.
Enzymatic activation sometimes removes many residues, such as about 200 residues from 505 residues when bovine carboxypeptidase B is activated.
(4) Excitatory proteins and inhibitory proteins
After binding to calcineurin (CAM), it can bind to many enzymes, activating it. An enzyme during the visual excitation process contains an inhibitory subunit, and when this subunit is reversibly released, the activity of the enzyme increases.
Second, the regulation of enzyme content
(1) Adjustment of synthesis speed
There is a class of enzymes called inducers, which are induced in cells by specific inducers. Its content is significantly increased in the presence of inducers. Inducers are generally their substrates or analogues. Other enzymes with essentially unchanged content are called structural enzymes. Inducerases are more common in microorganisms, such as galactosidase of E. coli, and the addition of lactose to the medium induces the production of lactose so that the bacteria can use lactose.
The distinction between structural enzymes and inducer enzymes is relative, only a difference in quantity, not an essential difference. The synthesis of enzymes is controlled by both genes and metabolites. Genes are the internal causes of the formation of enzymes, but the formation of enzymes is also regulated by metabolites, inducers can increase the amount of enzymes, enzyme products can also produce a repressive effect, so that the amount of enzyme production is greatly reduced. That is, metabolites can control the rate and amount of enzyme production.
(2) Control of degradation
Enzyme volume can also be regulated by accelerating or slowing down the degradation of enzyme molecules, such as when hungry, the rate of arginase degradation in the liver slows down, the amount of enzymes increases; acetyl-CoA carboxylase degradation is accelerated, and the amount of enzymes is reduced.
Exam points for this chapter
Explanation of the title of this chapter
Enzymes: Biological catalysts, almost all but a few RNA are proteins. The enzyme does not change the balance of the reaction, just
By reducing activation, the speed of the reaction can be accelerated.
Desprolinease protein (apoenzyme): The portion of the protein in the enzyme after removing organic or inorganic cofactors or cofunches that may be required for catalytic activity.
Holoenzyme: An enzyme with catalytic activity, including all essential subunits, auxiliaries, and other cofactors.
Enzyme Viability Unit (U, active unit): A measure of the enzyme viability unit. The 1961 International Enzymology Conference stipulates that 1 enzyme activity unit refers to the amount of enzymes that can transform 1 μmol of substrate within 1 minute under specific conditions (25oC, the other is the most appropriate condition), or the amount of enzymes that can transform 1 μmol of related groups in the substrate.
Specific activity: The number of micromoles per minute of substrate transformed at 25oC per milligram of enzyme protein. Specific viability is a measure of enzyme purity.
Activation energy: The energy required to convert all molecules in a 1mol reaction substrate from their state to an excessive state.
Active energy: The enzyme contains the substrate binding site and the amino acid residue part involved in the catalytic conversion of the substrate into the product. The active site is usually located in a fissure between the domains or subunits of the protein or in a depression on the surface of the protein, usually composed of some amino acid residues that are close together in three dimensions.
Acid-base catalysis: The catalytic effect of proton transfer to accelerate reactions.
Covalent catalysis: A substrate or part of a substrate forms a covalent bond with a catalyst and is then transferred to a second substrate. Many enzyme-catalyzed group transfer reactions are carried out by covalent methods.
Proximity effect: The increase in the speed of a non-enzymatic catalytic reaction or enzymatic reaction is due to the substrate's proximity to the active site, resulting in an increase in the effective concentration of the reactant at the active site, which will lead to more frequent formation of an over-state.
Initial velocity: The rate at which the substrate is converted into a product in the initial stage of the enzymatic reaction, the concentration of the product in this stage is very low, and its inverse reaction is negligible.
Michaelis-Mentent equation: The velocity equation representing the starting velocity (υ) of an enzymatic reaction vs. substrate concentration ([s]): υ=υmax[s]/(Km+[s])
Michaelis constant: For a given reaction, the concentration of the substrate at the starting rate (υ0) of the enzymatic reaction reaches half the maximum reaction rate (υmax).
Catalytic number (Kcat): Also known as a conversion number. is a kinetic constant, a measurement of how fast an enzyme (or an enzyme active site) catalyzes a reaction while the substrate is saturated. The catalytic constant is equal to the maximum reaction speed divided by the total enzyme concentration (υmax/[E]total). or the amount of substrate per second converted into products per molase active site (mole).
Double-reciprocal plot: That's called Lineweaver_Burk plotting. The reciprocal of the speed of an enzymatic reaction (1/V) versus the reciprocal of the substrate degree (1/LSF). The intercept on the x and y axes represents the reciprocal of the Michaelis constant and the maximum reaction velocity, respectively.
Competitive inhibition: A type of enzyme inhibition that can be reversed by increasing the concentration of the substrate. Competitive inhibitors usually compete with normal substrates or ligands for the binding site of the same protein. This inhibition increases km without υmax constant.
Noncompetitive inhibition: Inhibitors bind not only to free enzymes, but also to enzyme-substrate complexes. This inhibition makes Km constant and υmax smaller.
Uncompetitive inhibition: An enzymatic reaction inhibition in which an inhibitor binds only to an enzyme-substrate complex and not to a free enzyme. This inhibition makes both Km and υmax smaller but υmax/Km unchanged.
Serine protease: The active site contains proteins of serine residues that act as nucleophiles during catalysis.
Zymogen: By limited proteolysis, it is possible to change from inactive to a precursor of an enzyme with catalytic activity.
Regulatory enzyme: An enzyme located at a key site within one or more metabolic pathways whose activity increases or decreases according to metabolic needs.
Allosteric enzyme: An enzyme whose activity is regulated by binding to other molecules at sites other than the active site.
Allosteric modulator: Combined with a biomolecule that regulates the catalytic activity of the enzyme at the regulatory site of an allosteric regulatory enzyme, the allosteric modulator may be an activator or an inhibitor.
Concerted model: A pattern in which the same ligand is synergistically bound to oligopolymers, and according to the simplest homogeneous mode, the conformation of the protein is transformed between T (conformation with low affinity to substrates) and R (conformations with high affinity for substrates) due to the binding of a substrate or allosteric regulator. This model proposes that all protein subunits have the same conformation, either T conformation, or R conformation.
Sequential model: Another pattern in which the same ligand binds to oligopoly proteins in synergy. According to the simplest pattern of order change, the binding of a ligand induces a change in the tertiary structure of the subunit to which it binds, and causes great changes in the conformation of adjacent subunits. According to the order change pattern, only one subunit pair has a high affinity for the ligand.
Isoenzyme isozyme: A group of enzymes that catalyze the same chemical reaction and chemically form different enzymes. They differ from each other in terms of amino acid sequence, affinity of substrates, etc.
Allosteric modulator: This is called an allosteric effector. Binds to the modulating site of allosteric enzymes, modulates the catalytic activity of the enzyme biomolecules. Allosteric regulators may be activators or inhibitors.
Chapter VI Nucleic Acids
Nucleic acid classification Distribution and function
Two. nucleotide
Base Purines and PyrimidineS Nucleosides in DNA and RNA Polyphosphate Nucleotides Cyclic Nucleotides
3. The structure of DNA
Primary structure of phosphodiester bond DNA Secondary structure of DNA Tertiary structure of DNA Topology of DNA
IV. Structure of RNA
Differences between DNA and RNA Types and Functions Structure Characteristics of tRNAs Structural Characteristics of mRNA
Five. Physicochemical properties of nucleic acids
Denaturation and resensorption of UV-absorbed DNA Restriction enzymes
Section 1 Overview of a discovery
Nucleic acids, which account for 5-15% of the dry weight of cells, were discovered by the Swiss physician Miescher in 1868 and called "nuclides". For a long time, the "four-nucleotide hypothesis" was popular, believing that nucleic acids are composed of equal amounts of four nucleotides and could not have any important function.
In 1944, Oswald Avery demonstrated for the first time that DNA was genetic material through translational experiments by Streptococcus pneumoniae. Pneumococcus normal has a layer of viscous luminescent polysaccharide pods, which are pathogenic and called smooth (type S); a mutant type is called rough type (type R), without capsular membranes, and has no pathogenic capacity (lack of UDP-glucose dehydrogenase). In 1928, Griffith discovered that the transformation of Diplococcus pneumoniae, that is, the injection of live rough bacteria and heat-killed smooth bacteria into mice, can cause disease, and both alone are not pathogenic. This indicates that there is a substance in the body of the smooth bacteria killed by heating that converts the rough bacteria into smooth bacteria. Avery graded the cell-free extract of heat-killed smooth bacteria, and then determined the transformational activity of each component, and published a paper in 1944 stating that "dna ribosylated nucleic acid is the basic unit of the transformation elements of pneumococcal type".
The experimental evidence is as follows:
1. The analysis of the purified, highly active chemical elements of the transformed elements is very close to the calculated DNA composition.
2. The purified transformation elements are similar to DNA in optical, ultracentrifugation, diffusion, and electrophoresis properties.
3. Its transformational activity is not lost due to the extraction of proteins or lipids.
4. Treatment with trypsin and chymotrypsin does not affect its transformational activity.
5. Treatment with RNase does not affect its transformational activity.
6. DNase can lose its transformational activity.
After Avery's paper was published, some people still insisted that proteins were genetic material, believing that his isolation was incomplete and was a mixture of trace amounts of protein-induced transformation. In 1952, Hershey and Chase's T2 phage spin-cutting experiments thoroughly demonstrated that genetic material was nucleic acids, not proteins. They labeled proteins with 35S and nucleic acids with 32P. Infect the bacteria with the labeled phage and then determine the isotopic labeling of the host cells. When infected with sulfur-labeled phages, radioactivity is only present outside the cell, that is, on the shell of the phage; when a phosphorus-labeled phage is infected, the radioactivity is inside the cell, indicating that what enters the cell at the time of infection is DNA, and only DNA is continuous substance, so DNA is genetic material.
In 1956, Fraenkel Conrat's tobacco mosaic virus (TMV) reconstruction experiment proved that RNA could also be used as genetic material. The TMV is shaken in water and phenol to separate the protein from the RNA, and then infected with tobacco separately, and only the RNA can infect the tobacco and produce normal offspring.
The double helix structure model of DNA was established in 1953 and is considered one of the major breakthroughs in natural science of this century. In the following 30 years, nucleic acid research has won the Nobel Prize 15 times, accounting for 1/4 of the total, which shows the important position of nucleic acid research in life sciences.
Second, the classification of nucleic acids
Nucleic acids are macromolecules composed of nucleotides, the smallest molecular weight is the transport RNA, with a molecular weight of about 25kd; the molecular weight of human chromosomal DNA is as high as 1011kd. Nucleic acids are divided into DNA and RNA, DNA is mainly concentrated in the nucleus, and there are also small amounts of DNA in mitochondria and chloroplasts. RNA is mainly distributed in the cytoplasm. For viruses, either only DNA or only RNA. Therefore, viruses can be divided into DNA viruses and RNA viruses.
Nucleic acids can be divided into single strands (ss) and double strands (ds). DNA is generally double-stranded, as an information molecule; both RNA and single-stranded are present.
Section 2 Nucleotides I. Structure of Nucleotides
Nucleotides can be broken down into nucleosides and phosphoric acid, which in turn can be broken down into bases and pentose. Therefore, nucleotides are composed of three types of molecular fragments. There are two types of pentose, D-ribose and D-2-deoxyribose. Nucleic acids can therefore be divided into two categories: DNA and RNA.
(1) Base (base)
Bases in nucleic acids are divided into two categories: purines and pyrimidines.
1. Pyrimidine (pyrimidine, py) is a derivative of pyrimidine, there are three types: cytosine (cytosine, Cyt), uracil (uracil, Ura) and thymine (thymine, Thy). Uracil is only found in RNA and thymine is only found in DNA, but very small amounts of thymine are also found in some tRNAs. Cytosine is common to two types of nucleic acids, there is also 5-methylcytosine in plant DNA, and some Escherichia coli bacteriophage nucleic acids do not contain cytosine, but are replaced by 5-hydroxymethylcytosine. Because it is affected by the electron absorption effect of nitrogen atoms, the 2, 4 and 6 positions of pyrimidine are prone to substitution.
2. Purine alkali (purine, pu) is derived from purines, and there are two common types: adenine (adenine, Ade) and guanine (guanine, Gua). Purine molecules are close to planar, but slightly curved. There are also xanthine, hypoxanthine, uric acid, tealine, theobromine and caffeine in nature. The first three are metabolites of purine nucleotides, which are antioxidants, and the latter three are contained in plants and are methylated derivatives of xanthine, which have the effect of enhancing cardiac function.
In addition, some plant hormones, such as zeaxanthin, agonists, etc., are also purines, which can promote cell division and differentiation. Some antibiotics are purine derivatives. For example, purinemycin, which inhibits protein synthesis, is a derivative of adenine.
In organisms (A+T)/(G+C) it is called asymmetric ratios, which vary from organism to organism. For example, the asymmetric ratio of humans is 1.52, yeast is 79, and Garcinia cambogia is 0.35.
3. Rare bases In addition to the above five basic bases, there are also some rare bases with very little content in nucleic acids, most of which are methylated bases. Methylation occurs after the synthesis of nucleic acids and is of great significance for the biological function of nucleic acids. The methylated base content in nucleic acids generally does not exceed 5%, but can be as high as 10% in tRNA.
(b) Nucleosides
Nucleosides are formed by condensation of pentose and base groups. The first carbon atom of the sugar is connected to the first nitrogen atom of the pyrimidine or the ninth nitrogen atom of the purine with a glycosidic bond, generally referred to as an N-glycoside bond. Pentose is a furan ring, C1 is an asymmetric carbon atom, and glycosidal bonds in nucleic acids are all β glycosidic bonds. The base is perpendicular to the sugar ring plane. Glycosides are named by first saying the base name, followed by "nucleoside" or "deoxynucleoside".
In the tRNA contains a small amount of pseudouracil nucleoside (denoted by Ψ), which is connected to the C5 of the pyrimidine ring.
It is prescribed that the base is represented by a three-letter symbol, a single-letter symbol is used to represent nucleosides, and the addition of d in front of it indicates deoxynucleosides. The atoms of pentose are numbered with numbers with ', and bases are numbered with numbers without '.
(3) Nucleotides
The pentose hydroxyl group in nucleosides is esterified by phosphate to form nucleotides. There are three hydroxyl groups on the glycoside's sugar ring that form three nucleotides: 2', 3' and 5'-ribonucleotides. There are only two types of DNA: 3' and 5'. Most of the free presence in living organisms is 5' nucleotides. Hydrolyzing RNA with alkali gives a mixture of 2' and 3' ribonucleotides.
Rare bases can also form corresponding nucleotides. More than a dozen DNA have been found in natural DNA, and dozens of RNA have been found.
(4) Polyphosphate nucleotides
There are some free polyphosphate nucleotides within the cell, which have important physiological functions. 5'-NDP is the pyrophosphate of the nucleoside, and 5'-NTP is the triphosphate of the nucleoside. The most common are 5'-ADP and 5'-ATP. Phosphate residues on ATP are numbered from near to far in αβγ. When the outer two phosphate bonds are hydrolyzed, they can release 7.3 kcal of energy, while the ordinary phosphate bonds are only 2 kcal, so they are called high-energy phosphate bonds (~P). Therefore, ATP plays an extremely important role in cellular energy metabolism, and many chemical reactions need to be provided by ATP. High-energy phosphate bonds are unstable, and in 1NHCl, hydrolysis at 100 °C can be shedding in 7 minutes, while α phosphoric acid is much more stable. This property allows the determination of the content of unstable phosphorus in ATP and ADP.
Intracellular polyphosphate nucleotides often form complexes with magnesium ions. GTP, CTP, UTP also have the effect of transmitting energy in some biochemical reactions, but are not common. UDP can be used as a carrier to carry glucose in polysaccharide synthesis, and CDP as a carrier to carry choline in the synthesis of phospholipids. Various triphosphate nucleotides are precursors to the synthesis of DNA or RNA.
Guanine nucleoside tetraphosphate and pentaphosphate play a role in metabolic regulation, and in E. coli, they are involved in the control of rRNA synthesis.
(5) Cyclized nucleotides
Phosphoric acid forms an ester bond with two hydroxyl groups on the nucleoside at the same time, forming a cyclic nucleotide. The most common are 3',5'-cyclized adenylate (cAMP) and cGMP. They are the second messenger of the hormonal action and play a signaling role. It can be hydrolyzed by phosphodiesterase to form the corresponding 5'-nucleotide.
2. Relevant abbreviation symbols
Bases are represented by three-letter symbols, nucleosides are represented by capitalized single-letter symbols, and d is preceded by deoxynucleosides. The atoms of pentose are numbered with numbers with ', and bases are numbered with numbers without '.
Rare nucleosides (modified nucleosides) are also represented by a single-letter symbol, such as D for dihydroauracil nucleoside and T for thymidine. If there is a modified group on the base, add a lowercase letter representing the modified group before the uppercase letter representing the nucleoside, indicate the position of the modification in the upper right of this lowercase letter, and indicate the number of modified groups in the lower right (if only one can be omitted). For example, m2G represents 2-N-methylguanosine, m2,2,73G represents N2, N2,7-trimethylguanosine, and S4U represents 4-thiouridine. Modified groups on ribose are written to the right of the uppercase letters representing nucleosides, as Cm represents 2'-O-methylcytidine.
Nucleotides can be represented by lowercase p next to the nucleoside symbol, 5' nucleotides written on the left, and 3' nucleotides written on the right. Writing a few indicates a few phosphoric acids. 3',5'-Cyclized nucleotides can be preceded by lowercase c,2',3'-cyclized nucleotides can be followed by 〉P, such as U〉P for 2',3'-cyclic uridic acid.
Third, the function of nucleotides
1. As a component of nucleic acids.
2. Provide energy for energy-needing reactions. UTP is used for polysaccharide synthesis, GTP is used for protein synthesis, CTP is used for lipid synthesis, and ATP is used for multiple reactions.
3. For signal transmission. For example, cAMP and cGMP are the second messengers.
4. Participate in the composition of coenzymes. Such as NAD, FAD, CoA, etc. all contain AMP components.
5. Participate in metabolic regulation. Such as guanosine tetraphosphate can inhibit the synthesis of ribosomal RNA. Another example is antisense RNA.
Section 3 Structure of DNA
First, the primary structure of DNA
DNA is a polymerization of thousands of DNA. Its primary structure is the composition and arrangement of its components, that is, the sequence of bases.
2. Structure
In DNA molecules, adjacent nucleotides are bonded in 3',5'-phosphodiesters to form long chains, and the 3'-hydroxyl group of the former nucleotide binds to the 5'-phosphate of the latter nucleotide. The alternating arrangement of phosphoric acid and sugar in the chain constitutes the DNA phosphate backbone, and one end of the chain has a free 5'-phosphate group, called the 5' end; the other end has a free 3'-hydroxyl group, called the 3' end. In DNA, each dna ribose is linked to a base, and a specific sequence of bases carries genetic information.
3. Writing
When writing DNA, press from 5' to 3' from left to right, with 5' and 3' at the end of the chain, such as 5'pApGpCpTOH3'. The middle phosphoric acid can also be omitted and written as pAGCT. This is a literal abbreviation. There are also linear abbreviations, with vertical lines for pentose, 1' on top and 5' on bottom.
Second, the secondary structure of DNA
(1) Establishment of double helix structure
The elucidation of the structure of the DNA double helix is one of the most significant natural science achievements of this century. In the 1940s, it had been found that dehydrated DNA had a high density, and X-ray diffraction showed that there were periodic structures of 0.34 nm and 3.4 nm in the DNA. In 1950, Chargaff discovered the law of complementary pairing through the analysis of bases: in any DNA, A = T, G = C, so there is A + G = T + C.
In 1953, Watson and Crick established a double helix model of DNA based on Wilkins' DNA X-ray diffraction data and base composition, thus opening the prelude to modern molecular biology. At the time, Watson was only 24 years old, studying in the Cavendish Laboratory in Cambridge, and he recognized the importance of nucleic acids when he was in the United States, so he devoted himself to nucleic acid research when everyone was studying proteins, thus obtaining epoch-making results.
What Watson and Crick elucidated was a structural model of B-DNA crystallization that was broadly consistent with the DNA present within the cell. In recent years, it has been discovered that local DNA can also exist in the form of other double or triple helixes.
(2) The main points of the B-DNA double helix structure
1. Basic structure
The DNA double helix is a right-hand double helix composed of two inverted, parallel, and complementary STRANDs of DNA. The DNA phosphate skeleton of the two chains is reversed and paralleled according to the right-hand spiral, spiraling around a common axis on the outside of the double helix, and the bases of the two chains are paired one by one, arranged in parallel in the center of the double helix, and the base plane is perpendicular to the axis. The two strands in the DNA double helix are complementary to each other.
2. Basic data
The double helix has an outer diameter of 2 nm and a pitch of 3.4 nm, rising one turn per 10 pairs of bases. Therefore, the distance of each pair of bases is 0.34 nm, and the angle is 36 degrees.
3. Force
There are two forces that stabilize the structure of the double helix. In the horizontal direction are the hydrogen bonds between the paired bases, the A=T pair forms two hydrogen bonds, and the GC pair forms three hydrogen bonds. These hydrogen bonds are the main force that overcomes the repulsion of the phosphate group between the two chains and binds the two chains to each other. In the vertical direction, it is the accumulation force between the bases and planes. The accumulation force is the common embodiment of hydrophobic force and van der Waals force. Both hydrogen bonds and accumulation forces are themselves a synergistic interaction, and there are also synergies between the two.
4. Large and small ditches
The DNA phosphate skeleton does not completely enclose the base pair, and on the surface of the double helix there are two furrows that coincide with the direction of the double helix, one is deeper and wider, called the large ditch; and the other is narrower and shallower, called the small groove. The side of the large ditch is exposed to C6, N7 and C4, C5 of the purine and the pyrimidine and their substituent groups; the side of the small ditch is exposed to the C2 of the purine and the C2 of the pyrimidine and their substituent groups. Therefore, the structural characteristics of base pairs can be identified from the two grooves, and various enzymes and protein factors can identify the characteristic sequences of DNA.
5. Correction
The above model is the average characteristic of DNA, which varies locally due to the influence of the base sequence. For example, the angle between two nucleotides can vary from 28 degrees to 42 degrees, and there is a certain angle between the complementary paired bases, called propeller-like twisting. The spiral circle contains 10.4 base pairs.
(3) Other structures of DNA
DNA fibers form B-DNA at 92% relative humidity. DNA sodium, potassium or calcium salts can form an A-type structure at 75% relative humidity, which is also a right-hand spiral, but the bases are slightly inclined (19 degrees), and the pitch and skeleton structure are slightly different, with 11 base pairs per turn, short and thick. Its biological significance lies in its close resemblance to the conformation of double-stranded RNA and DNA-RNA hybrids in solution. Due to the presence of 2'-hydroxyl groups, RNA is not easy to take a B-type conformation, so when transcribed, DNA should take a type A conformation. Some organic solvents and proteins can turn B-type DNA into A-type conformations.
C-shaped structures were found in DNA lithium salt fibers with 66% relative humidity. It can be assumed that the C-type conformation occurs in concentrated salt solutions and glycol solutions. At this time, the accumulation force decreases, and the hydrogen bonding energy is relatively increased. The C-type structure is also a right-hand spiral, present in chromatin and certain viruses. There are also D-shapes and two metastable structures called T and P. DNA regions rich in A-T pairs have a large structural diversity.
DNA also has a left-handed helix, or Z-DNA. Its skeleton is serrated, found in oligomerIC DNA alternating between purines and pyrimidine, and is also an anti-parallel complementary double helix with 12 base pairs per turn, and the spiral is elongated. This shows that the base sequence of DNA stores not only genetic information, but also information about its own advanced structure. Z-DNA, as a special structural marker, is associated with the regulation of gene expression.
Third, the tertiary structure of DNA
(1) Super-spiral
The tertiary structure of DNA refers to the further distortion of the double helix. Its basic form is the superhelix, that is, the spiral of the spiral. The tertiary structure is determined by the secondary structure. B-DNA is coiled at every 10 bases with the lowest energy and is in a stretched state; when the discs are more or less rounded, tension occurs, forming a superhel. A positive (right-hand) superhelix is formed when the disc goes around for a long time, and a negative superhel when it is insufficient. Because the superhelix is formed under the tension of the double helix, only the double-stranded closed loop DNA and the linear DNA fixed at both ends can form the superhelix, and the DNA with the incision cannot form the superhel. Whether it is the double-stranded linear DNA of eukaryotes or the double-stranded ring DNA of prokaryotes, it exists in the form of a negative superhelix in vivo, and the density is generally 100-200bp a circle. The formation of a negative hyperhelix in DNA is a structural and functional imperative.
(2) Dynamic changes in the high-level structure
The higher structure of DNA within cells is variable. Through the action of multiple protein factors and enzymes, changing the secondary and tertiary structure of DNA is a need for biological function. DNA replication, transcription, recombination, and repair are all accompanied by changes in its advanced structure. In living organisms, there are three ways to change the higher structure:
Under the action of destreating enzymes, the hydrogen bonds between the base pairs are destroyed, so that the dna is partially unchained into a single-stranded region to increase the number of coils of the unsolved double-stranded region, thereby increasing the positive superhelix or reducing the negative supercoil.
By locally forming a Z-DNA (left-handed) double helix, the number of coils in the B-DNA part can also be increased, reducing the negative superhelix.
One or two strands of DNA are cut off by topoisomerases, and under the impetus of the double helix tension, the broken end is rotated around the complementary chain, and the tension is released before the connection, which can eliminate the superhelix and also introduce the supercoil.
The topology of DNA has the following formula:
L=T+W
where L is called the number of chains, which is the number of times a chain is wound around another chain in a right-handed spiral, which must be an integer. The winding number T is the double helix cycle and W is the superhex number. T, W can be decimal. The supercoil density is generally between -0.03 and -0.09.
(3) The multi-level nature of the supercoil
The chromosomes of eukaryotes are complexes of DNA and proteins, where the hyperhelix structure of DNA is multi-layered. Chromosomes are formed by multiple curls of chromatin filaments. Chromatin filaments are beaded by nucleosome repeating units. Nucleosomes are made up of DNA and histone proteins. Histones are alkaline proteins rich in arginine and lysine, and there are 5 kinds of H1, H2A, H2B, H3 and H4. The last four protein cores with 2 molecules each forming the nucleosome, and about 140 bp double helix DNA (core DNA) orbits 1.75 times outside the protein core, which together constitute the core particles of the nucleosome. There is about 60 bp of junction DNA between the core particles. 1 Molecular histone H1 binds to the entry and exit sites of the junction DNA, fixing the core DNA to the periphery of the core protein. The nucleosome is oblate spherical, about 6 nm high and 11 nm in diameter. The nucleosome repeat unit consisting of core DNA and junction DNA comprises about 200 bp and is compressed from 68 nm to 11 nm in length. So the first superhelix turned a 2nm diameter DNA double helix into a chromatin filament with a diameter of 11nm, and the length was compressed by 6-7 times. The chromosomal filaments are once again superhelixed to form a chromosomal coarse filament with a diameter of 30 nm, and the length is compressed by 6 times. The third super-helix coils the coarse wire into a unit fiber with a diameter of 400 nm and compresses the length by 40 times. Finally, the unit fibers are folded to form chromatids, and the length is compressed by 4-5 times. Thus, after 4 hyper-helixes, the length of the DNA is compressed by nearly 10,000 times (8400 times).
Section 4 Structure of RNA
First, the structural characteristics of RNA
1. The RNA molecule is a single strand. It can be folded back and paired with each other to form a hairpin or stem ring structure. A local A helix must be formed with at least 4-6 base pairs. Some molecules can account for 50% of the folding.
2. The ribose in the RNA molecule has a 2'-hydroxyl group, but it is not used for bonding.
3. Ureamine instead of thymine, containing a variety of rare bases.
4. RNA is a transcription product of the sequence of parts of DNA with a much smaller molecular weight. Some viruses contain RNA replication enzymes that can catalyze RNA synthesis with RNA as a template, i.e. RNA replication.
5. RNA is multi-copy.
6.RNA is divided into three categories according to function: transport RNA (tRNA), messenger RNA (mRNA), and ribosome RNA (rRNA). There are also snRNAs and hnRNAs. The former is related to the processing of RNA, and the latter is a precursor to mRNA.
2. Transport RNA
(1) Primary structure
Transporter RNAs are small molecules, generally composed of 74-93 nucleotides, with a molecular weight of around 25kd and a sedimentation coefficient of 4s. Its function is to transport amino acids and synthesize proteins according to the base sequence of messenger RNA. Each of the 20 amino acids has a specific transporter RNA, and some have 2 or more transporter RNAs. There are 30-40 tRNAs for prokaryotes and 50-60 or more for eukaryotes. There are reports that there is an RNA (tRNASer) that specifically transports selenoidine and recognizes UGA (stop code).
tRNA is the nucleic acid with the most modified components. Of the about 70 modified components that have been discovered, 50 are present in the tRNA. Each tRNA molecule has a modified component, some as many as a dozen, accounting for 20% of the total building blocks. Modified ingredients include modified bases and modified nucleosides, both of which are modified by processing 4 standard bases or nucleosides after transcription. In tRNA molecules, the modified base is mainly methylated bases, and the modified nucleosides are mainly pseudouracil nucleosides.
(b) Secondary structure of tRNA
Single-stranded RNA borrows partial sequences to complement each other and can form a definite secondary structure. The forces that maintain the secondary structure are also hydrogen bonds and accumulation forces. The basic unit of the secondary structure of the RNA molecule is the hairpin structure. The RNA strand folds itself, two complementary sequences are paired to form a double helix, and the unpaired bases between the two segments form a protrusion ring. The hairpin structure is formed by a double helix and a loop. The folding ratio is high and the structure is stable.
The tRNA molecules all have a clover-shaped secondary structure consisting of an arm and three hairpins. The double helix region of the 5' and 3' end sequences of the tRNA strand is called the amino acid arm, and the 3' end of the tRNA strand has an unchanged single-stranded CCAMOH, named because the terminal A binds to the amino acid. The three hairpins consist of a dihydrouracil loop with a DHU stem, an anticodon ring and an anticodon stem, a TψC ring and a TψC stem. The three bases in the center of the anticodon ring form the anticodon, which is paired with the codon of the messenger RNA. Some tRNAs have an additional variable stem of varying lengths between the anticodon stem and the TψC stem.
(iii) Tertiary structure of tRNA
The tRNA molecule is further distorted on the basis of the secondary structure to form a determined tertiary structure. The tertiary structure of the various tRNAs resembles an inverted L. At the upper right end of the molecule is the amino acid arm, and at the lower end is the anticodon. The distance between the two ends is about 8 nm. The fine structure of different tRNAs is different and can be recognized by the specific amino acid tRNA ligase and related protein factors.
3. Ribosomal RNA
Higher animal ribosomes have 4 rRNA components: 18s, 28s, 5.8s, and 5s, which together with more than 80 proteins form the ribosomals of eukaryotes (80s). Ribosomes can be broken down into two subunits of size, small subunits (40s) consisting of 18s rRNA and 33 proteins, and large subunits consisting of 28s, 5s, 5.8s rRNA and 49 proteins. Prokaryote ribosomes (70s) consist of three rRNAs and more than 50 proteins, large subunits (50s) include 23s, 5s rRNA and 34 proteins, and small subunits (30s) include 16s rRNA and 21 proteins.
The primary structure of various rRNAs has been measured. rRNA contains only a small number of modified components, mainly methylated nucleotides, including modified bases such as m7G and m6G and various 2'-O-methyl modified nucleosides.
The secondary structure of the allogeneic rRNA has common characteristics.
4. Messenger RNA
As a template to guide the synthesis of proteins, mRNA has the characteristics of many types, few copies, short lifespan, and few modified components. The main sequence of the mRNA is a coding region with non-coding regions on both the 5' side and downstream of it. Eukaryote mRNA molecules also have 5' caps and 3' tail structures at each end. Prokaryotic cells generally do not have tails, and some eukaryotic viruses do.
The simplest hat structure is the reversed 7-methylguanosine triphosphate, which is connected to the original 5' terminal nucleotide of the mRNA by 5' ppp5' to form m7GpppN. The more complex cap structure is also modified in the back of one or both nucleotides as well as 2'-O-methyl. The general form of the hat structure can be written as m7GpppN(m)pN(m).... The cap structure is important for stabilizing mRNA and its translation, it encloses the 5' terminal and protects it from the hydrolysis of nucleic acid exonucleases, and can also be used as a recognition signal of the protein synthesis system, which is recognized by a specific protein factor to initiate the translation process.
The 5' non-coding region is a shorter sequence between the hat and the coded region starting codon, which includes the sequence of the sign translation starting, such as the SD sequence of prokaryotes. The coding region starts from the starting codon AUG and ends with the terminating codon (UAG, UGA, UAA), encoding the primary structure of a protein. Each of the three bases forms a codon, encoding an amino acid. The 3' side non-encoded region is the transcription sequence after the stop codon, which includes a sequence of AAUAAA, which may be a sign of adding a 3' tail, or a coordinated signal of translation termination. The 3' end tail is a multipoly A tail. Mature mRNA generally has a polyA tail of 20-200 bases at its 3' end, which is a marker of the nuclear membrane pore transport system and is related to the transport of mature mRNA through the nuclear membrane pores to the cytoplasm.
Section 5 Physicochemical Properties of Nucleic Acids
First, viscosity
Macromolecular solutions are viscosity larger than ordinary solutions, and linear macromolecules are more viscous than spherical macromolecules. DNA is a linear macromolecule, the total amount of human diploid DNA is 3.3×109 bp, the total length can reach 1.75 meters, the average length of DNA molecules is more than 4 cm, while the double helix diameter is only 2 nm, and the ratio of length to diameter is as high as 107. Therefore, the DNA viscosity is extremely high and it is also very easy to break under mechanical force. When the double-stranded DNA is desprosioned into single-stranded DNA, it changes from a more extended double helix to a more compact line structure, and the viscosity decreases significantly. RNA has a much lower viscosity because of its small molecular weight and linear group structure.
Second, density
The buoyancy density of macromolecules can be determined using density gradient centrifugation. CsCl has a large solubility and can be made into 8M solution. The buoyancy density of DNA is generally above 1.7, RNA is 1.6, and protein is 1.35-1.40. The sedimentation coefficients of DNA with different molecular weights and structures are different, and the ratio of sedimentation coefficients of linear double helix DNA, linear single-strandED DNA, and superhel DNA is 1:1.14:1.4. Therefore, by measuring the sedimentation coefficient, the structure of DNA and its changes can be understood.
Third, ultraviolet absorption
Purines and pyrimidines are strongly absorbed by ultraviolet because of their conjugated systems. Nucleic acids have UV absorption peaks at 260 nm and proteins at 280 nm. The concentration and purity of nucleic acids can be determined by UV absorption. OD260/OD280 is generally measured, with DNA =1.8 and RNA =2.0. If it contains protein impurities, the ratio is significantly reduced. Impure nucleic acids cannot be determined by UV absorption. UV absorption changes are a sign of changes in DNA structure, when the double-stranded DNA is desproducted, the base exposure increases, and the UV absorption increases significantly, called the color enhancement effect. The ratio of UV absorption of double-stranded and single-stranded DNA to nucleotides is 1:1.37:1.6.
Fourth, dna degeneration
Under certain conditions, the phenomenon of double-stranded DNA unchained into single-stranded DNA is called denaturation or melting. Heat-induced denaturation is called thermal denaturation; under alkaline conditions (pH>11.3), DNA becomes alkaline. In addition, urea, organic solvents, and even desalination can cause DNA denaturation. After removing the denaturing factor, the complementary single-stranded DNA can be re-bound to double-stranded DNA, called renatural or annealed. DNA recombination begins with a slow nucleation reaction in which local sequences are paired to form a double-stranded core, and then completed by a rapid so-called zipper reaction.
After DNA denaturation, the viscosity decreases, and the density and absorbance increase.
The process of denaturing single-stranded DNA binding to a DNA strand or RNA molecule with the same sequence to form a double-stranded DNA-DNA or DNA-RNA hybridization molecule is called hybridization or molecular hybridization. The development and application of molecular hybridization technology has played an important role in promoting the development of molecular biology and biological high technology.
The temperature at which 50% of the DNA molecule is denatured is usually referred to as the melting point (Tm). In general, the melting point of DNA under physiological conditions is between 85-95 degrees. The melting point depends mainly on the base composition, and the higher the G-C pair content, the higher the melting point. Generally, the melting point of G-C pairs with a content of 40% is 87 degrees, and for each 1% increase, the melting point increases by about 0.4 degrees. Ionic strength also has an impact, because ions can bind to DNA to stabilize it, so the lower the ion strength, the lower the melting point, and the narrower the melting range. Therefore DNA should be kept in a highly salt solution. If the DNA is impure, the denaturation temperature range also expands. Formamide can make the hydrogen bond between base pairs unstable, reducing the melting point. Therefore, formamide is often used to denature DNA in molecular biological experiments to avoid DNA breakage caused by high temperatures. Ethanol, acetone, urea, etc. can also promote DNA denaturation.
The rate of re-regrowth of DNA is related to its initial concentration of C0 and its complexity. When other conditions such as temperature and ionic strength are fixed, the C0t value of half of the DNA recombination is only related to its complexity, which can be used to calculate the complexity of the genome.
5. Restriction enzymes
Restriction enzyme II recognizes and cleaves specific palindromic sequences, which organisms use to prevent the effects of foreign DNA, and is used in genetic engineering for the cutting of DNA, known as molecular scalpels. For example, EcoRI, E is the genus name, co is the species name, R is the strain name, and I is the discovery order.
Sixth, the purification of nucleic acids
Extraction: Generally, the cells are broken first to obtain DNP or RNP. Then remove the protein with phenol-chloroform, precipitate the nucleic acid with ethanol or isopropanol, dry it and then dissolve it.
Purification: Commonly used by electrophoresis or chromatography. PAGE is generally used to separate nucleic acids below 1K, such as sequencing. Larger ones should be electrophoretic with agarose. Purification of mRNAs is commonly used in olego-dT chromatography columns or magnetic beads.
After the 1980s, nucleic acid research has the following characteristics:
1. RNA research is valued. Previous research focused on DNA, and now RNA is a research hotspot. The discovery of ribase and the processing and editing mechanism of RNA are two major discoveries. A gene is transcribed from different transcription initiation sites in different tissues or different physiological states, and different proteins can be formed through different splicing methods and different 3' terminal maturation mechanisms, which is a more flexible regulatory mode than gene rearrangement. The application of RNA is also increasingly widespread, such as cutting viral nucleic acids with ribozyme and blocking the expression of harmful genes with antisense RNA. Therefore, some people call the 90s a decade of RNA.
2. Research materials from prokaryotic to eukaryotic. The replication, transcription, and translation of eukaryotes are much more complex than prokaryotics, and the change of materials has led to major discoveries such as ribozyme, RNA splicing, editing, etc., which have greatly promoted the research of nucleic acids.
3. Study the interaction of nucleic acids with nucleic acids, nucleic acids with other biological macromolecules. Most of the nucleic acids in the organism are in various complexes, and their structure and function are related to the complex. Research on transcriptional regulation of eukaryotic genes has focused on cis-acting elements, trans-acting factors, and interactions between them. The structure and function of ribosomes and the synthesis of aminoyl tRNAs have always been two important objects for studying the interaction between nucleic acids and proteins, and recently spliceo-some, nuclear heterogeneous ribonucleoproteosomes (hn-RNPs), nuclear small molecule ribonucleoproteins (snRNPs), editors (editosome) and other research hotspots have been formed.
The study enters a phase where the molecular level is combined with the overall level. Rather than if the development of flies is controlled by a network of regulated genes, some laboratories are conducting studies in a way that combines the whole with the molecular level.
Nucleoside :is a compound composed of purine or pyrimidine bases that are joined to pentose by covalent bonds. Ribose and base are generally linked by the β-N- sugar bond formed between the isocephalic carbon of the sugar and the N-1 of the pyrimidine or the N-9 of the purine.
Uncleoside: A compound formed by hydroxyl phosphorylation in the pentose component of a nucleoside.
cAMP (cycle AMP): 3ˊ,5ˊ-cyclic adenylate, is the second messenger within the cell, due to some hormone or other molecular signal stimulation to activate adenylate cyclase catalyzed ATP cyclization.
phosphodiester linkage: A chemical group that refers to two ester bonds formed by the esterification of one molecule of phosphoric acid with two alcohols (hydroxyl groups). The ester bond forms a bridge between the two alcohols. For example, the 3ˊ hydroxyl group of a nucleoside is phosphorylated with the 5ˊ hydroxyl group of another nucleoside with the same molecule, forming a disureabide bond.
Deoxyribonucleic Acid (DNA): A polydeoxynucleotide containing a special DNA sequence that is connected between the DNA through 3ˊ,5ˊ-phosphate disalith bonds. DNA is the carrier of genetic information.
Ribonucleic acid (RNA): A polyribonucleotide of a special ribonucleotide sequence formed by a 3ˊ,5ˊ-phosphate disalicyclic bond junction.
Ribonucleic acid (RRNA): As a class of RNA components, rRNA is the richest RNA in cells.
Messenger ribonucleic acid :a class of RNA used as templates for protein synthesis.
Transfer ribonucleic acid: A class of RNA that carries activated amino acids, brings them to protein synthesis sites and integrates them into growing peptide chains of RNA. TRNAs contain anti-codes that recognize complementary codes on the template mRNA.
Transformation: The effect of a foreign DNA being introduced into a host bacterium through some means, causing changes in the genetic characteristics of the bacterium.
Transduction: With the help of viral vectors, genetic information is transferred from one cell to another.
Base pair: Two nucleotides in a nucleic acid chain paired by hydrogen bonds between bases, such as A and T or U, and G and C paired.
Chargaff's rules: the molar content of adenine and thymine in all DNA is equal (A=T), and the molar content of guanine and cytosine is equal (G=C), which is equal (A+G=T+C). The base composition of DNA has species specificity, but not tissue and organ specificity. In addition, changes in the trophic state and environment during the growth and developmental stages do not affect the base composition of DNA.
DNAdouble helix of DNA: A conformation of nucleic acid in which two reverse parallel chains of polynucleothizic acid are wound around each other to form a right-handed double helix. The base is located on the inside of the double helix, and the phosphoric acid is on the outside with the glycosyl group, which is connected by the phosphate disalidin bond to form the skeleton of the nucleic acid. The base plane is perpendicular to the center axis of the illusion, the sugar ring plane is parallel to the axis, and both chains are right-handed spirals. The diameter of the double helix is 2nm, the base accumulation distance is 0.34nm, the angle between the two nucleoglyclic acids is 36 ゚, and each pair of helixes is composed of 10 pairs of bases, and the bases are paired and complementary by A-T and G-C, and they are linked to each other by hydrogen bonds. The force that maintains the stability of the DNA double helix structure is mainly the base accumulation force. The surface of the double helix has two large grooves of varying width and narrowness and depth, and a small ditch.
Large groove and small groove: Spiral grooves (grooves) that appear on the surface of the B-DNA double helix, wide grooves called large grooves, narrow grooves called small grooves. Large furrows, small furrows, are caused by base pair accumulation and torsion of the sugar-phosphate backbone.
The curling of DNA superhelix (DNAsupercoiling) :D NA itself is generally the result of the bending understring (negative superhelix) or overswing (positive superhelic) of the DNA double'helix.
Topoisomerse: An enzyme that changes the number of DNA chains by cutting off the phosphodiester bonds in one or two strands of DNA and then rewinding and sealing them. Topoisomerase I, reducing the negative superhelix by cutting off a strand in dna, increasing the number of consecutive rings. Certain topoisomerase II is also known as DNA spinase.
Nucleosome: The structural unit used to package chromatin, it is made up of a strand of DNA wrapped around a histone nucleus.
Chromatin: an amorphous substance that exists in the nucleus of eukaryotes and is easily colored by alkaline dyes. Chromatin contains intact double-stranded DNA that acts as a backbone, as well as histone' non-histone and a small amount of DNA.
Chromosome: A form of genetic material with a fixed form that is formed by tightly wound 'folding' condensation and fine packaging of chromatin during cell division. Simply put, a chromosome is a large single, double-stranded DNA molecule composed of a complex of related proteins that contain many genes that store and transmit genetic information.
DNA denaturation (DNAdenaturation) :D the phenomenon of NA double-strand unchaining and separating into two single strands.
Annealing: The process by which DNA changes from single-stranded recombination to a double-stranded structure. Single strands of DNA of the same origin are completely restored to the structure of the double strands after annealing, and hybridization molecules are formed between homologous DNA ' between DNA and RNA, after annealing.
Melting temperature (Tm): The midpoint temperature of the temperature range at which the two-stranded DNA is melted completely into a single-stranded DNA.
Hyperchromic effect: When double helix DNA is melted (unchained), ultraviolet absorption increases at 260 nm.
Hypochromic effect: A phenomenon in which ultraviolet absorption decreases as nucleic acids recur.
Exonuclease: Enzymes in ribonucleases and dnaases that hydrolyze phosphodiester bonds within nucleic acid molecules.
Exonuclease: An enzyme that hydrolyzes nucleolicide one by one from one end of the nucleic acid chain.
Restriction endonuclease: An endonuclease that hydrolyzes double-stranded DNA at a special nucleoglycide sequence. Type I restriction enzymes can catalyze both the methylation of host DNA and the hydrolysis of non-methylated DNA, while type II restriction enzymes only catalyze the hydrolysis of non-methylated DNA.
Restriction map: The same DNA is cleaved with different restriction enzymes to obtain the cleavage sites of various restriction enzymes, and the resulting site map is established to facilitate the analysis of the structure of the DNA.
Inverted repeat sequence: A repeating nucleotide sequence that exists in the opposite direction within the same polynucleotide. Reverse duplication in double-stranded DNA may cause the formation of cross-shaped structures.
Recombination DNA technology: Also known as genomic engineering, a technique that uses restriction enzymes and vectors to recombine a certain gene of interest and vector DNA of one organism and then transfer it to another organism's cells for replication and expression according to pre-designed requirements.
Gene : also known as cistron , refers to a fragment of DNA that is transcribed. In some cases, genes are often used to refer to a fragment of DNA that encodes a functional protein or DNA molecule.