chemical nature of enzymes
Enzymes are biocatalysts that are synthesized by cells and perform catalytic functions in the body.
Reactions catalyzed by enzymes are called enzymatic reactions
substrate
Substances that undergo chemical reactions catalyzed by enzymes
product
Substances produced by enzymatic reactions
chemical nature
Mainly protein, a few RNA
Classification of enzymes
simple enzyme
Enzymes that are pure proteins
conjugation enzyme
In addition to proteins, certain non-amino acid components (cofactors) are bound
Cofactor
coenzyme
Small organic molecules that are loosely bound to apoenzyme and can be removed by dialysis
prosthetic base
Small organic molecules that are covalently bound to enzyme proteins and tightly bound to apoenzymes and cannot be removed by dialysis or ultrafiltration.
catalytic properties of enzymes
Common properties of enzymes and non-enzyme catalysts
Can only catalyze reactions that are thermodynamically allowed
After the reaction is completed, it is not consumed or changed and can be reused.
The catalytic effect on forward and reverse reactions is the same
The equilibrium constant is not changed, but the speed to equilibrium is accelerated or the time to equilibrium is shortened.
unique catalytic properties of enzymes
efficiency, specificity
absolute specificity
Acts only on one substrate and not on any other substances
relative specificity
Can act on a class of structurally similar substrates
Three theories explaining enzyme specificity
Three Points and One Line Doctrine
Distinguish enantiomers and explain stereospecificity
Binds to substrate at active center
active center
The region of an enzyme molecule that directly binds to the substrate and is directly related to catalysis
Mostly hydrophobic aa residues, a small amount of hydrophilic aa residues (the catalytic reaction is hydrophilic aa)
The active center is structurally complementary to the substrate, and the conformation is not fixed.
Mild reaction conditions, sensitive to reaction conditions, easy to inactivate
Factors affecting enzymatic reactions
enzymatic reaction rate
The change in concentration of substrate or product per unit time
Influencing factors
ionic strength
Metal ion concentration
Can serve as a cofactor for enzymes, thereby affecting the reaction rate
Reaction medium pH
Affects the dissociation state of enzyme molecules
Affects the dissociation state of a substrate, prosthetic group or coenzyme
enzyme activators or enzyme inhibitors
enzyme concentration
Under certain pH and temperature conditions, when the substrate concentration is much greater than the enzyme concentration, the reaction rate is directly proportional to the enzyme concentration.
substrate concentration
Third-order reaction with substrate concentration
When the substrate concentration is low, a first-order reaction occurs
As the substrate concentration increases, the reaction behaves as a mixed-level reaction
When the substrate concentration is quite high, a zero-order reaction occurs.
enzyme substrate intermediate theory
Michaelis reaction kinetics
Describe the relationship between Michaelis enzyme reaction rate and substrate concentration
Derivation of Michaelis-Menten equation
The reaction rate is the initial rate
The enzyme-substrate complex is in steady state, that is, the ES concentration does not change.
According to the law of mass action
At steady state, the formation rate of ES is equal to the dissociation rate of ES
Interpret the Michaelis-Menten equation
Km is the substrate concentration at which the initial rate of the enzyme reaction is half of Vm
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 concentration of the enzyme.
Under certain conditions, it can reflect the affinity between the enzyme and the substrate. The greater the Km, the lower the affinity between the enzyme and the substrate.
When the Km value of the enzyme for the substrate is less than the Km value of the product, the reaction is conducive to the forward reaction, otherwise it is conducive to the reverse reaction.
Vmax
Theoretical maximum reaction rate, which changes with enzyme concentration
Kcat
The catalytic constant or conversion number or turnover number of an enzyme specifically refers to the total number of molecules that an enzyme molecule converts a substrate into a product per unit time.
kcat=k2=Vmax/Et
Can measure the speed of an enzymatic reaction
Kcat/Km
It is used to measure the catalytic efficiency of an enzyme and can also reflect the perfection of an enzyme.
The dual nature of the Michaelis-Menten equation
When the substrate concentration is very low, that is, S is much smaller than Km, the Michaelis-Menten equation changes to
Comply with first-order kinetics
When the substrate concentration is very high, that is, S is much greater than Km, the Michaelis-Menten equation can be transformed into
Compliant with zero-order dynamics
Double reciprocal plot of Michaelis enzyme
Enzyme inhibitor effect
reversible inhibitor
Reversibly binds to enzyme via secondary bond
competitive inhibitor
Inhibitors compete with substrate
noncompetitive inhibitor
Can be combined with both ES and E
anticompetitive inhibitor
Can only bind to ES, so that the substrate bound to the active center can no longer be converted into products
irreversible inhibitor
Irreversibly binds to the enzyme through strong chemical bonds and is irreversible once inactivated.
gene specific inhibitors
Covalent modification of essential side chain groups on the enzyme active center
substrate analog inhibitor
Structurally similar to the substrate, it binds to the enzyme at the active center and irreversibly modifies the essential groups on the active center of the enzyme.
Transition state analog inhibitors
Very similar to the transition state of an enzymatic reaction, it binds to the active center of the enzyme, causing the substrate to be unable to bind to the enzyme.
suicide inhibitor
Catalyzed by an enzyme, several reactions occur, but no product is formed. Instead, essential groups of the enzyme are modified, resulting in loss of enzyme activity.
Kinetics of allosteric enzymes
allosteric effect
After the non-catalytic part of the enzyme molecule is reversibly and non-covalently combined with certain compounds, the conformation changes, thereby changing the active state of the enzyme, which is called the allosteric effect of the enzyme.
Enzymes with specific effects are called allosteric enzymes
Substances that can cause allosteric effects on enzyme molecules are called allosteric effectors
Properties of allosteric enzymes
rate, substrate concentration curve is S-shaped
Bidirectional responses to competitive inhibition
At low concentrations, it induces a positive synergistic effect of allosteric enzymes and increases the reaction rate of the substrate.
At high concentrations, it occupies the active center of the enzyme, and the substrate cannot combine with S normally, inhibiting the reaction rate.
Mild denaturation can lead to loss of allosteric effects
Usually oligomeric enzymes, and allosteric enzymes account for a minority
Hill equation
h=1, it is not an allosteric enzyme and has no substrate cooperativity.
h>1, the plot of rate versus substrate concentration is S-shaped, with positive substrate cooperativity.
S-curve
Enzymes are sensitive to changes in substrate concentration in the environment and sensitively regulate metabolism.
h<1, the enzyme has negative substrate cooperativity
apparent hyperbola
The enzyme is extremely insensitive to changes in substrate concentration in the environment, ensuring that important reactions are not affected by the substrate and can always proceed.
enzyme catalytic mechanism
Contents of transition state stability theory
In the process of any chemical reaction, the reactants need to reach a specific high-energy state to react. This unstable high-energy state is called a transition state. To reach the transition state, the reactants must have enough energy to break through the activation energy.
The affinity of the enzyme to the transition state is much higher than the affinity to the substrate (ground state), and the catalytic effect of the enzyme comes from the stabilization of the transition.
Chemical mechanism of transition state stabilization
proximity directed response
It means that two or more substrates are combined with the enzyme at the active center of the enzyme at the same time, freezing the translation and rotation of certain chemical bonds, so that they adopt the correct spatial orientation, greatly increasing the concentration of the substrates
Generalized acid-base catalysis
Refers to the amino acid residues involved in catalysis in the active center of the enzyme gaining or losing protons (transferring to transition state intermediates to achieve the effect of stabilizing the transition state.). This mechanism is involved in the catalytic process of most enzymes.
If the pKa value is close to 7, the side chain group may be the most effective generalized acid-base catalyst, such as the imidazole group of His residue. Under physiological conditions, half is acid and half is base, and the rate of releasing protons is fast.
Reaction rate depends on pH and buffer concentration
electrostatic catalysis
The distribution of active center charges is used to stabilize the transition state of an enzymatic reaction. The enzyme uses its own charged group to neutralize the opposite charge generated when a reaction transition state is formed to catalyze it.
Sometimes the substrate is introduced into the active center of the enzyme through the electrostatic interaction between the enzyme and the substrate.
metal catalysis
metalloenzymes
Contains tightly bound metal ions
metal activating enzyme
loosely bound to metal ions in solution
How metal ions catalyze
Functions as a Lewis acid
Binds to negatively charged substrates to promote correct orientation of the substrate during the reaction
Participate in electrostatic catalysis and stabilize negatively charged transition states
Participate in redox reactions as an electron donor or electron acceptor through reversible changes in valence state
as a component of enzyme structure
covalent catalysis
During the catalytic process, the enzyme temporarily forms unstable covalent intermediates with certain groups on the substrate.
nucleophile
A reagent that donates a pair of electrons to a reactant to form a covalent bond
electrophile
A reagent that obtains a pair of electrons from a reactant to form a covalent bond
Typically, Lewis acids are electrophiles and Lewis bases are nucleophiles.
Substrate deformation
When the enzyme meets the substrate, the enzyme induces the sensitive bonds in the substrate to become more sensitive, thereby generating electronic tension and deformation, bringing the substrate closer to its transition state.
Structure and function of several common enzymes
serine protease
The catalytic group includes an indispensable serine residue, and DIFP is its irreversible catalyst
trypsin
Has a deep substrate binding pocket, suitable for binding long-chain Lys and Arg
chymotrypsin
The substrate binding pocket is wide, and the pocket wall is distributed with hydrophobic amino acids, which is particularly suitable for the binding of aromatic amino acids.
elastase
Shallow binding pocket, suitable for amino acids with smaller side chains (such as glycine)
catalytic triad
Ser195
Affinity group that acts as an attacking substrate
His57
As a generalized acid-base catalyst
Asp102
Target His57 and affect its pH to change its acid-base properties
Oxygen anion hole
Stabilization of tetrahedral transition states by hydrogen bonding
Summary of serine protease catalytic process
Ser195 generalized acid catalysis, covalent catalysis (attack substrate)
His57 generalized acid-base catalysis
Two pro-nuclear attacks
Ser-O attacks the carbonyl group of the substrate to form a covalent bond (tetrahedral transition state)
H2O-OH attacks the carbonyl group of the covalent complex to form a covalent bond (tetrahedral stable state)
an oxygen anion hole
Stabilization of tetrahedral transition states by hydrogen bonding
Metalloproteinase
There are metal ions bound to the active center, and their activity absolutely requires metal ions, so the metal chelating agent EDTA can cause the loss of its activity.
aspartic protease
The catalytic group includes two aspartates, which are inactive under alkaline pH conditions.
Thiol protease
The catalytic group includes a cysteine residue and iodoacetic acid is its irreversible catalyst
Regulation of enzyme activity
Quantitative changes of enzymes (controlling enzyme levels, concentrations)
Control the expression of enzyme genes and the degradation of enzyme molecules
isoenzyme
Enzymes that catalyze the same reaction but have different structural properties
Example: Four isoenzymes of hexokinase
HK1
Mainly distributed in the brain
HK2
Mainly distributed in skeletal muscles
HK3
Mainly distributed in white blood cells
HK4
Mainly distributed in the liver (glucokinase)
Different tissue distribution and different concentration changes respond to the different needs of the body
hexokinase
inhibited by product feedback
Control the speed of glycolysis
Glucokinase
Monomeric enzyme, not subject to allosteric inhibition
Responsible for lowering blood sugar and maintaining stability
Qualitative change of enzyme (controlling enzyme activity)
Modulate existing enzyme activity without changing enzyme concentration (low energy consumption, fast)
allosteric adjustment
In addition to the active center, some allosteric enzymes also contain allosteric centers that can bind to some special ligand molecules, thereby changing the conformation of the enzyme and causing changes in enzyme activity.
Both synergistic and non-synergistic enzymes can be allosterically regulated
covalent modification
Enzyme molecules are reversibly covalently modified under the catalysis of other enzymes. This enzyme is called a covalent modification regulatory enzyme. It is a key enzyme that regulates some metabolisms. It is regulated by hormones and can be cascade amplified.
Some enzymes take the modified state as the active state
Some enzymes are active in a demodified state
Several forms of covalent modification of enzymes
Characteristics of covalent modification regulation
Enzymes exist in two differently modified or differently active forms
There are changes in covalent bonds
Influenced by other regulatory factors (e.g. hormones)
Generally an energy-consuming process
There is an amplification effect
hydrolytic activation
The process of converting inactive enzymes (zymogens) into active enzymes under the catalysis of enzymes
activation mechanism
Under the catalysis of enzyme, the excess peptide fragments of the zymogen are cut off
The essence of activation
Form the active center of the enzyme
physiological significance
A method of biological self-protection
A way to regulate enzyme activity to ensure normal metabolism in the body
Activation or inhibition of regulatory proteins
Certain proteins act as ligands to bind to specific enzymes and regulate the activity of the bound enzyme.
This protein is called a regulatory protein
Polymerization and dissociation of enzyme subunits
Characteristics of zymogen activation
The process is accompanied by changes in the primary structure of the enzyme protein
Hydrolysis process, irreversible, one-time adjustment
It is a rapid signal amplification process achieved through the cascade effect to complete specific functions.
PS: Catalytic mechanism of lysozyme
Generalized acid-base catalysis
electrostatic catalysis
Asp52 (negative charge) stabilizes positively charged transition state substrates
Substrate deformation
In order to adapt to the conformation of the active center of the enzyme, the sugar chain changes from chair to half-chair, making the glycosidic bond more likely to break.
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