Chapter 3 Enzyme1 introduction to enzymes

Chapter 3 Enzyme1 introduction to enzymes

Lecture Outline Introduction to enzyme
Properties and catalytic mechanisms of enzymes Structure and function of enzymes Nomenclature and classification of enzyme Kinetics of enzyme-catalyzed reaction Regulation of enzyme activity Clinical application of enzymes

Essential Question: What are enzymes, and what do they do?
Living Cells: Thousands of chemical reactions are proceeding very rapidly at any given instant. All reactions are catalyzed by special biocatalysts, enzymes —protein and occasionally RNA. Reactions in cells are different from general chemical reactions: easily, rapidly regulated and controlled Enzyme-catalyzed reaction take place usually under relatively mild conditions.

What do enzymes do? Enzymes are biological catalysts that accelerate the rates of chemical reactions. Snail with enzyme catalyst Snail without enzyme catalyst

What is the difference between an enzyme and a protein?
RNA Enzymes All enzymes are proteins except some RNAs and not all proteins are enzymes

Substrates, products, and enzymes
Enzymes catalyze the rate at which substrates are converted to product Substrates Product Enzyme

Enzymes catalyze the conversion of substrates into products
What is a substrate? A substrate is the compound that is converted into the product in an enzyme catalyzed reaction. For the reaction catalyzed by aldolase, fructose 1,6-phosphate is the substrate.

What is the difference between enzyme catalyzed reactions and uncatalyzed chemical reactions?
Enzyme catalyzed reactions are much faster than uncatalyzed reactions. Enzyme catalyzed reactions display saturation kinetics with respect to substrate concentration. Enzyme catalyzed reactions are optimized for specific temperature and pH values.

Molecular components Simple enzymes: enzymes require no other chemical groups other than their amino acid residues for activity. consists of only one peptide chain. trypsin, chymotrypsin, ribonuclease A Conjugated enzymes: enzymes contain chemical groups other than AA, the non-amino acid parts are usually called cofactors the protein part alone called apoprotein (apoenzyme). holoenzyme= apoenzyme (pr) +cofactor (non-pr)

Many vitamins, organic nutrients required in small amounts in the diet, are precursors of cofactors.
Cofactors: metal ions, small molecules, Cofactors are divided two groups according to the binding ability. ----coenzyme -----prosthetic group

Coenzymes: Loosely bind to apoenzyme. Be able to be separated with dialysis Accepting H+ or group and leaving to transfer it to others, or vise versa. Prosthetic groups: Tightly bind through either covalent or many non-covalent interactions. Remained bound to the apoenzyme during course of reaction.

Metal ions Metal-activated enzyme: ions necessary but loosely bound. Often found in metal-activated enzyme. Metal ion is essential for activity Metalloenzymes: ions tightly bound Particularly in the active center, transfer electrons, bridge the enzyme and substrates, stabilize enzyme conformation, neutralize the anions. Metal ion is retained throughout purification

Organic componds Small size and chemically stable compounds
Transferring electrons, protons and other groups Vitamin-like or vitamin-containing molecule Coenzymes often function as transient carriers of specific (functional) groups during catalysis.

Monomeric\oligomeric\multienzyme enzymes
Monomeric enzyme: only contain a polypeptide chain with tertiary structure. Oligomeric enzyme: contain two or more polypeptide chains associated by noncovalent forces. Multienzyme complex: different enzymes catalyzed sequential reactions in the same pathway are bound together. Multifunctional enzyme(tandem enzyme): a single polypeptide chain with multiple activities

Components of enzyme Essential groups: all groups essential for maintaining the enzyme activity active center: a specific region of the enzyme that contains some chemical groups for binding substrates and transforming it into products.( some functional groups are close enough in space to form a portion) The active site is a three-dimensional conformation formed by groups that come from different parts of the linear amino acid sequence. Look like a cleft or a crevice. hydrophobic.

Two essential groups Active site contains
binding groups (specificity of binding): to associate with the reactants to form an enzyme-substrate complex determine specificity for substrate catalytic groups (participate in the catalytic processes) :to catalyze the reactions and convert substrates into products determine characteristics of catalyzation

Other essential groups
Maintain conformation of enzyme Substrate Other essential groups Ser: OH Cys: SH His: imidazole Catalytic groups Binding groups Active site

Essential groups of the active site

In many enzymes, the active site has shape complementary to those of their substrates only after the substrates are bound (the induced fit). For conjugated enzymes, the active site always contains coenzymes.

Two models have been proposed for substrate and enzyme binding:
(1) Lock and key model (2) Induced fit model

Emil Ficher: (1894) Dan Koshland: (1958)
The ‘Lock & Key’ hypothesis - explains substrate specificity - says nothing about why catalysis occurs Dan Koshland: (1958) ‘Induced Fit’ hypothesis: - enzymes prefer to bind to a distortion of the substrate that resembles the transition state - both enzyme and substrate must adjust to one another - in reality, enzyme is not ‘distorted’ but has evolved to bind in a certain way and sometimes undergo conformational changes

Lock and key model

Induced fit model

An Example: Induced conformational change in hexokinase
Catalyzes phosphorylation of glucose to glucose 6-phosphate during glycolysis such a large change in a protein’s conformation is not unusual BUT: not all enzymes undergo such large changes in conformation

Advantage of the induced fit mechanism
The active site can be open to allow substrates to bind, then close over the substrates to provide optimum transition state stabilization Disadvantage of the induced fit mechanism Energy that would otherwise be used to help stabilize the transition state of the reaction Energy must be used to induce the conformational change in the enzyme.

Notice that in both the Lock and Key and Induced Fit models, the active site is designed to stabilize the transition state of the reaction.

Several factors contribute to enzyme catalysis
Proximity and orientation effects Electrostatic effects Acid-base catalysis Covalent catalysis

What characteristic features define enzymes?
Enzymes are remarkably versatile biochemical catalysts that have in common three distinctive features: catalytic power, specificity, and regulation Common features: 2 “do” and 2 “don’t Unique features: 3 “high”

Common features Do not consume themselves: no changes in quantity and quality before and after the reactions Do not change the equilibrium points: only enhance the reaction rates. Apply to the thermodynamically allowable reactions Reduce the activation energy

Unique features Enzyme-catalyzed reactions have very high catalytic efficiency. Enzyme have a high degree of specificity for their substrates. Enzymatic activities are highly regulated in response to the external changes.

High Catalytic power: the ratio of E-catalyzed rate of a reaction to the uncatalyzed rate
Enzyme display enormous catalytic power, accelerating reaction rates as much as 1016 over uncatalyzed levels. Urease is a good example: Catalyzed rate: 3x104/sec Uncatalyzed rate: 3x /sec Ratio is 1x1014 !

High Specificity: refers to the ability of an enzyme to discriminate between two competing substrates and catalyze one specific reaction Specificity: the selective qualities of an enzyme (selecting substrate) Based on structural complementarity, enzyme recognize substrate. Unlike conventional catalysts, enzymes demonstrate the ability to distinguish different substrates.

Enzymes are highly specific both in the reaction catalyzed and in their choice of substrates.
An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions. There are three types of substrate specificities. Absolute specificity Relative specificity Stereospecificity

Absolute specificity Enzymes can recognize only one type of substrate and implement their catalytic functions . For example, urease only hydrolyzes urea to form NH3 and CO2. not methylurea.

Relative specificity Enzymes catalyze one class of substrates or one kind of chemical bond in the same type Protein kinase A ,C,G, To phospharylate the –OH group of serine and threonine in the substrate proteins, leading to the activation of proteins.

stereospecificity The enzyme can act on only one form of isomers of the substrates. Lactate dehydrogenase can recognize only the L-form but the D-form lactate.

High regulation Enzyme-catalyzed reactions can be regulated in response to the external stimuli, satisfying the needs of biological processes Regulation can be accomplished through varying the enzyme quantity, adjusting the enzymatic activity, or changing the substrate concentration.

Classification of enzyme
1956, International Commission on Enzymes (IUBMB) -Classified by the types of reactions that they catalyze 6 classes recognize Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases With each class subdivided into further subclasses

(1) Oxidoreductase Catalyze oxidation-reduction reactions.
catalyze the transfer of hydrogen atoms and electrons Contains dehydrogenase and oxidase. For example: lactate dehydrogenase, peroxidase.

(2) Transferase Catalyze transfers of groups between donors and acceptors. For example: glutamate-pyruvate trasaminase (GPT), acyltransferase.

(3) Hydrolase Catalyze cleavage of bonds by addition of water.
For example: pyrophosphatase, peptidase.

(4) Lyase Catalyze lysis of a substrate, generating a double bond or adding a substrate to a double bond of a second substrate. For example: pyruvate decarboxylase, aldolase.

(5) Isomerase Catalyze racemization of optical or geometric isomers and certain intramolecular oxidation-reduction reactions. For example: alanine racemase, mutase.

(6) Ligase or Synthetase
Join two molecules at the expense of a high energy phosphate bond of ATP. For example: glutamine synthetase, carboxylase.

Enzyme nomenclature the suffix –ase : urease, phosphatase,
Traditional nomenclature: the suffix –ase : urease, phosphatase, some bearing little resemblance to their activity: trypsin and pepsin (proteases) — easy confuse Systematic nomenclature: Each enzyme is given a systematic name which identifies the reaction catalyzed. Hexokinase----ATP: D-hexose-6- phosphotrasferase

A four digital number can precisely identify all enzymes
Each enzyme is assigned a four-digit number with the first digit denoting the class it belongs, the other three further clarifications on the reaction catalyzed. Hexokinase: E.C class 2, transferase

Common name: Lactate dehydrogenase (LDH)
Systematic name Substrates are stated first, followed by the reaction type to which the ending-ase is affixed. Serial number: EC. X.X.X.X （ EC——Enzymes Commission ） Lactate:NAD+ oxidoreductase EC Major class----oxidoreductase Subclass----oxidation group CHOH Subsubclass----NAD+ as hydrogen acceptor No. in this subsubclass Common name: Lactate dehydrogenase (LDH)

Some definitions Apoenzyme = the protein part of an enzyme without coenzymes or prosthetic groups that are required for the enzyme to have activity. (Note: many enzymes do not have coenzymes or prosthetic groups bound to them). Coenzyme = small organic or inorganic molecules which are bound to the apoenzyme and are required for the enzyme to catalyze the chemical reaction. Prosthetic group = similar to a coenzyme, but is tightly bound to the apoenzyme. Heme is a prosthetic group in cytochrome c and hemoglobin. Holoenzyme = the apoenzyme with the coenzyme or prosthetic group bound to it (i.e. the active form of the enzyme).

Enzyme 2 kinetics

Kinetics：is a study on the rate of enzyme-catalyzed reactions and the factors affected the reaction rate. The factors affected the rate of enzyme-catalyzed reactions： pH activator inhibitor substrate enzyme temperature

For a reaction to occur, the substrates must first overcome an energy barrier. The higher the energy barrier, the slower the reaction will occur..

For most of the reactions in our bodies, the substrates must overcome large energy barriers for the reaction to occur. This prevents us from spontaneously bursting into flames. Enzymes lower the energy barrier for a reaction, speeding up the reaction in a controlled fashion and preventing undesirable side reactions from occurring.

Enzymes catalyze reactions by lowering their activation energies
. . Uncatalyzed Enzymes stabilize the transition states of reactions ) Reaction DG Enzyme Catalyzed Reaction Free Energy ( Substrates D G rxn Products Reaction Coordinate

Intermediate state Forming an enzyme-substrate complex, a transition state, is a key step in the catalytic reaction Transition state is a fleeting molecular moment (not a chemical species with any significant stability) that has the highest free energy during a reaction. The combination of a substrate and an enzyme creates a new reaction pathway whose transition state energy is lower than that of the reaction in the absence of enzyme. Decrease the activation energy

Initial velocity Enzyme activity is commonly expressed by the initial velocity ( V0) of the reaction being catalyzed. The reaction rate is defined as the product formation per unit time. The slope of product concentration ([P]) against the time in a graphic representation is called initial velocity. It is of rectangular hyperbolic shape.

Reaction velocity curve
Vi rectangular hyperbola

First order reaction: The rate of the reaction is directly proportional to [S] only when [S] is low.
Zero-order reaction: When [S] is sufficiently high, the velocity approach maximum velocity (Vmax).

Michaelis-Menten Equation
The mathematical expression of the product formation with respect to the experimental parameters Michaelis-Menten equation describes the relationship between the reaction rate and substrate concentration [S]

Effect of substrate At early times in the reaction, the concentration of the product ([P]) is negligible and the overall reaction can be written as (no reverse reaction) k k3 E + S  ES  E + P k2

Michaelis-Menten Equation (1913)
Km： Michaelis constant K2+ K3 Km= K1

[S] K V + = m Vm 当 [S] << Km时, V = [S] Km 当 [S] >> Km时,
V ≌ Vm

Understanding Km Km is a constant derived from rate constants
Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S, because at equilibrium, Reversible reaction, dissociation constant is So small Km means tight substrate binding; high Km means weak substrate binding. Km equals to the substrate concentration at which v=1/2vmax k1 k-1

Km has units of molarity. Km indicates the affinity of E and S.
Km is characteristic constant of an enzyme-catalyzed reaction. Km has units of molarity. Km indicates the affinity of E and S.

Km is independent of [E]
Km is independent of [E]. It is determined by the structure of E, the substrate and environmental conditions ( ph, T, ionic strength,) Km is a characteristic constant of E. The value of Km quantifies the affinity of the enzyme and the substrate. The larger the Km, the smaller the affinity.

The theoretical maximal velocity
Understanding Vmax The theoretical maximal velocity Vmax is a constant Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate Vmax is asymptotically approached as substrate is increased The reaction velocity of an enzymatic reaction when the binding sites of E are saturated with substrates. It is proportional to [E].

The dual nature of the Michaelis-Menten equation
Combination of 0-order and 1st-order kinetics When S is low, the equation for rate is 1st order in S When S is high, the equation for rate is 0-order in S The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!

A measure of catalytic activity
The turnover number A measure of catalytic activity kcat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. If the M-M model fits, k2 = kcat = Vmax/Et Values of kcat range from less than 1/sec to many millions per sec It can be used to compare catalytic activity of enzymes.

Enzyme Units are used to define the activity of an enzyme
IU (international unit) ----International Commission on Enzymes One IU of enzyme: the amount that catalyzes the formation of 1 micromole of product in 1 minute. One katal is that amount of enzyme catalyzing the conversion of 1 mole of substrate to product in 1 second. 1 katal equals 6 ×107 IU Specific activity: enzyme units per mg protein (Units/mg)

Linear Plots can be derived from the Michaelis-Menten Equation
Vmax[S] Km+ [S] = Vmax[S] Km+ [S] = 1 v = Km Vmax [S] 1 + Lineweaver-Burk double-reciprocal plot To determine Km and Vmax To identify the reversible repression y = mx + b

Factors affecting enzyme-catalyzed reaction
Substrate concentration Enzyme concentration Temperature pH Inhibitors activators

Effect of enzyme [E] affects the rate of enzyme-catalyzed reactions
[S] is held constant. When [S]>>[E], V=[E]

Effect of T Optimal T : T at which an enzyme has the maximal catalytic power. 35℃-40℃ for warm blood species . Reaction rates increase by 2 folds for every 10℃ rise. Higher T will denature the enzyme. It is not a characteristic constant of an enzyme Bell-shaped curve Enzyme activity optimum temperature Temperature ( °C )

Effect of pH Optimal pH at which the enzyme has the maximal catalytic power. pH 7.0 is suitable for most enzymes Particular examples: Ph (pepsin)= 1.8 Ph (trypsin)=7.8

inhibitors Inhibitors are certain molecules that can decrease the catalytic rate of an enzyme-catalyzed reaction. Inhibitors can be normal body metabolites and foreign substances (drugs and toxins)

Enzyme Inhibition Enzyme can be inhibited by inhibitors. Inhibitors are tools for scientists to understand enzymes. Inhibitors are also in many cases pharmaceutical reagents against diseases; Inhibitors inhibit enzyme function by binding with enzymes. The binding reaction can be either reversible or irreversible; Reversible inhibitors associate with enzymes through non-covalent interactions. Reversible inhibitors include three kinds: Competitive inhibitors; Non-competitive inhibitors; Un-competitive inhibitors Irreversible inhibitors associate with enzymes through covalent interactions. Thus the consequences of irreversible inhibitors is to decrease in the concentration of active enzymes.

Irreversible inhibition
Inhibitors are covalently bound to the essential groups of enzymes. Inhibitors cannot be removed with simple dialysis or super-filtration. Binding can cause a partial loss or complete loss of the enzymatic activity. 3 different types: Group Specific Reagent: - inhibitor does not resemble substrate Substrate Analogue: - inhibitor resembles substrate Suicide Inhibitors: - inhibitor resembles substrate, turns "dangerous" after processed by enzyme

+ E Example 1 organophosphorous compounds RO O HX P HO R’O X PAM
Organophosphorous compound: pesticides Acetylcholine esterase Inhibited AchE Acid Regeneration of active enzyme ——pyridine aldoxime methyliodide (PAM) PAM E Inhibited AchE active AchE Inactive inhibitor

Example 2 compounds with heavy metal ions
Hg2+ \ Ag+ \ As 3+ lewisite Inhibited enzyme -SH-enzyme Heavy metal containing chemicals bind to the –SH groups to inactivate the enzymes Regeneration of active enzyme ----2,3- dimercaptopropanol (BAL) BAL Inhibited enzyme Active enzyme Compond with As

Example 3 Antibiotic penicillin
Penicillin is a suicide inhibitor Resemble substrate Binds at enzyme active site (not an "inhibitor" yet) Processed by enzyme via normal catalytic mechanism Becomes a chemically active intermediates that modifies and inactivates the enzyme irreversibly Good candidate for drug due to minimal side effect Prevent the synthesis of the bacterial cell wall, killing the cell

Reversible inhibition
Inhibitors are bound to enzymes non-covalently The reversible inhibition is characterized by an equilibrium between free enzymes and inhibitor-bound enzymes. competitive inhibition non-competitive inhibition uncompetitive inhibition

Competitive inhibition
Competitive inhibition - inhibitor (I) binds only to E, not to ES

Competitive inhibitors share the structural similarities with that of substrates.
Competitive inhibitors competes for the active sites with the normal substrates. Inhibition depends on the affinity of enzymes and the ratio of [E] to [S] At high substrate concentration, the effect of a competitive inhibitor can be overcome.

Competitive Inhibitors
k1 K-1 k2 Km increases vmax unchanged k3 k-3 +inhibitor v 1/[S] 1/v 1/vmax -1/Km Slope=Km/vmax vmax Km(1+[I]/KI) [S] Km -1/(Km(1+[I]/KI))

vi 1 ′ Vmax is not affected. Km is increased.

Inhibition feature As [S] increases, the effect of inhibitors is reduced, leading no change in Vmax Due to the competition for the binding sites, Km rises, equivalent to the reduction of the affinity.

example FH4( tetrahydrofolate) is a coenzyme in the nucleic acid synthesis, and FH2(dihydrofolate) is the precursor of FH4. Bacteria cannot absorb FH4(folic acid )directly from environment. Bacteria use p-amino-benzoic acid (PABA), Glu and dihydropterin to synthesize FH2. but human body cannot one was infected by bacterium he could taken sulfa drug such as sulfanilamide Sulfanilamide derivatives share the structural similarity with PABA, blocking the FH2 formation as a competitive inhibitor. Bacteria would take sulfanilamide to synthesize its FH4, but the product is useless. Finally bacteria would be killed because of lack of normal FH4. because human body is unable to synthesize FH4, there is no effect when taking the drug.

Clinical application of competitive inhibition
Competitive inhibitors succinate fumeric acid malonate oxaloacetic acid malic acid Succinate dehydrogenase sulfanilamide p-amio-benzoic acid(PABA) dihydropterin glutamic acid PABA Dihydrofolate synthetase dihydrofolate (FH2) tetrahydrofolate (FH4)

Noncompetitive inhibition
Inhibitors bind to other sites rather than the active sites on the free enzymes or the E-S complexes. The E-I complex formation does not affect the binding of substrates. The E-I-S complexes do not proceed to form products Conformational changed Reducing the [ E-S] Vmax, reduce, unchanged Km.

The effect of a noncompetitive inhibitor cannot be overcome at high substrate concentrations.

Noncompetitive Inhibitors
k1 k2 k-1 KI’ KI Km unchanged vmax decreases +inhibitor 1/[S] 1/v 1/vmax -1/Km Slope=Km/vmax (1+[I]/KI)/Vmax Slope= Km(1+[I]/KI)/vmax v [S] vmax Km Vmax/(1+[I]/KI)

Uncompetitive inhibition
Uncompetitive inhibitors bind only to the enzyme-substrate complexes. The E-I-S complexes do not proceed to form products. The E-I-S complexes do not backward to the substrates and enzymes. This inhibition has the effects on reducing both Vmax and Km Commonly in the multiple substrate reactions

Uncompetitive Inhibitors
k1 k2 k-1 Km decreases vmax decreases Slope unchanged KI’ +inhibitor v 1/v vmax Slope=Km/vmax (1+ KI/[I])/Vmax Slope= Km/vmax Vmax/(1+KI/[I]) -1/Km 1/vmax [S] 1/[S] Km/(1+ KI/[I]) Km - (1+ KI/[I])/Km

Summary of Classes of Inhibitors
Competitive inhibition - inhibitor (I) binds only to E, not to ES Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition. Mixed inhibition-when the dissociation constants of (I) to E and ES are different. The inhibition is mixed.

Competitive Inhibitor Noncompetitive Inhibitor Uncompetitive Inhibitor
Inhibitor Type Binding Site on Enzyme Kinetic effect Vmax unchanged Km increased. Competitive Inhibitor Specifically at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium-like process. Inhibition is reversible by substrate. Noncompetitive Inhibitor Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate. Km unaltered Vmax decreased Binds only to ES complexes at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate. Vmax decreased Km decreased. Uncompetitive Inhibitor

activators Activator: substances enable non-active enzyme to become active one or low-active enzyme to become high-active one. Metals such as Mg2+, K+, Mn2+, etc. essential activator: enable non-active enzyme to become active one . For example, Mg2+ for hexokinase. non-essential activator : enable low-active enzyme to become high-active one . For example, Cl- for salivary amylase.

Draw a Lineweaver-Burk plot if Vmax=100 mmol/mL, Km=2mM.
Sample Questions What is the v/Vmax ratio when [S]=5Km? Draw a Lineweaver-Burk plot if Vmax=100 mmol/mL, Km=2mM. Draw the new lineweaver-Burk plot on the same plot as above if a competitive inhibitor is added. [I]=0.5 mM, KI=1mM.

Enzyme 3 regulation of enzyme

Enzymes need to be active in the right place at the right time
Inappropriate expression can lead to uncontrolled growth or cell (and organism) death

Many biological processes take place at a specific time; at a specific location and at a specific speed. The catalytic capacity is the product of the enzyme concentration and their intrinsic catalytic efficiency The key step of this process is to regulate either the enzymatic activity or the enzyme quantity.

Reasons for regulation
Maintenance of an ordered state in a timely fashion and without wasting resources Conservation of energy to consume just enough nutrients Rapid adjustment in response to environmental changes.

How do cells control specific biochemical reactions?
Control of enzyme activity Control of enzyme levels Control of enzyme location Control of substrate availability Removal or conversion of reaction products

What Factors Influence Enzymatic Activity?
The number of enzyme molecules The activity of enzyme molecules Enzyme Regulation Genetic regulation of enzyme synthesis and decay determines the amount of enzyme present at any moment. The availability of substrates and cofactors usually determines how fast the reaction goes. As product accumulates, the apparent rate of the enzymatic reaction will decrease. Enzyme activity can be regulated allosterically. Enzyme activity can be regulated through covalent modification. Modulator proteins regulate enzymes through reversible binding Regulation of enzyme activity also can be accomplished in other ways.

Enzyme activity regulation
The thousands of enzyme-catalyzed chemical reactions in living cells are organized into a series of biochemical or metabolic pathways. Metabolic and other processes are controlled by altering the quantity or the catalytic efficiency of key enzymes. Enzyme activity regulation allosteric regulation covalent modification Zymogen and activation of zymogen isoenzymes Enzyme amount regulation induction and repression (genetic control) enzyme degradation

Regulation of enzyme activity
allosteric regulation covalent modification Zymogen and activation of zymogen isoenzymes

Allosteric regulation
Allosteric enzymes are those whose activity can be adjusted by reversible, non-covalent binding of a specific modulator to the regulatory sites, specific sites on the surface of enzymes. Allosteric enzymes are normally composed of multiple subunits which can be either identical or different. The multiple subunits are Catalytic subunits: Where active sites are located. Substrate binds here Regulatory subunits: Effectors bind here Allosteric regulation: Metabolites binds to region of outside the active site, and change the conformation, and thus the enzyme activity.

Allosteric regulators do not bind to the active site of the enzyme
Activation or inhibition of an enzyme’s activity due to binding of an activator or inhibitor at a site that is distinct from the active site of the enzyme.

Allosteric enzymes: some definitions
1. Allosteric = “other site” other than active site 2. Regulatory molecules called, effectors, modulators, regulatory molecules 3. Homotropic regulation: regulation by substrate at active site 4. Heterotropic regulation: regulation by molecule NOT substrate ( end products), at allosteric site 5. Few enzymes are allosteric 6. Allosteric enzymes DO NOT exhibit M-M kinetics kinetic plot of V versus [S] is sigmoidal shape. Allosteric regulation can be positive or negative

Properties of regulatory enzymes
Their kinetics do not obey the Michaelis-Menten equation. S-shaped / hyperbolic curve In the jargon of allostery, substrate binding is cooperative. The regulatory effect by product is allosteric inhibition. Regulatory or allosteric enzymes are regulated by activation. Allosteric enzymes have an oligomeric organization. The regulatory effects are achieved by conformational changes occurring in the protein when effector metabolites bind..

Allosteric regulators shift the substrate
dependence curve In the above plot, the allosteric activator decreases the Km of the enzyme, while the allosteric inhibitor increases the Km of the enzyme

Allosteric activators stabilize the high affinity state of the enzyme

Allosteric inhibitors stabilize the low affinity state of the enzyme

Physiology of allosteric enzymes
Consider biochemical pathways: -Homotropic regulation, substrate activation activation E1 E2 E E E5 A B C D E F -Heterotropic regulation, end product inhibition E E E3 E4 E5 A B C D E F inhibition F inhibits C->D partially inhibits A -> B D E F A B C G H I

Feedback inhibition Feedback inhibition, i.e., building up of a pathway’s end product ultimately slows the entire pathway, is often realized through allosteric enzymes. The enzyme catalyzing the first step of a synthetic pathway is often an allosteric enzyme.

Concerted Model + cooperativity Assumes 2 conformation states: R & T
Binding of substrate induces all subunits to change to R state. No T-R hybrids. Allows for + cooperativity only. T state R state

Sequential Model The binding of substrate switches conformation of only the subunit to which it is bound. Conformational change in one subunit may  or  the affinity of other subunits have for the substrate. Allows for + or - cooperativity.

Sequential Model T T R T R R R R R R T T T T T T T R R R

Covalent modification
Covalent modification: Some groups of an enzyme can bind to certain chemical groups by covalent bond and change the enzyme activity. A variety of chemical groups on enzymes could be modified in a reversible and covalent manner. Such modification can lead to the changes of the enzymatic activity. The activity of many enzymes are regulated by reversible covalent modifications. Phosphorylation/dephosphorylation, the most common reversible covalent modification, is a highly effective means of switching the activity of target enzymes.

Common modification Phosphorylation- dephosphorylation
adenylation - deadenylation methylation demethylation uridylation deuridylation ribosylation deribosylation acetylation deacetylation

The most common modification is the addition and removal of a phosphate group: phosphorylation and dephosphorylation respectively. Protein kinases catalyze the transfer of a phosphate group from an ATP molecule to the side chains of Ser, Thr, or Tyr residues in proteins. Protein phosphatases catalyze the hydrolysis of phosphoryl groups attached to proteins, thus reversing the effects of kinases.

Protein kinases Phosphorylation introduces a bulky group bearing two negative charges, causing conformational changes that alter the target protein’s function Pi Classification of protein kinases Class I: Ser/Thr protein kinases, PKA, PKC, MAPK, CaM-PhK Class II: Ser/Thr/Tyr protein kinases Class III: Tyr protein kinases ATP, manganese ion

Features of covalent modification
Two active forms (high and low) Covalent modification Energy needed Amplification cascade Some enzymes can be controlled by allosteric and covalent modification. Reversible, usually requires one enzyme for activation and another for inactivation

zymogen zymogen: Some enzymes are synthesized as larger inactive precursors called proenzymes or zymogens. Zymogen activation: Zymogens are activated by the irreversible hydrolysis of one or more peptide bonds under certain conditions. The process of zymogen activation is actually the process of active site formation.

Mechanism of zymogen activation
Selective proteolysis Formation active center Conformational change zymogen Definite condition Examples include the hormone insulin (proinsulin), pepsinogen, trypsinogen, etc.

Regulation of digestive enzymes

A-peptide B-peptide C-peptide cut C-peptide proinsulin insulin

Pepsinogen is converted to pepsin by autocatalytic proteolysis at pH 2

plasma glucose homeostasis
Enzymes involved in protein digestion, blood clotting, and tissue and bone remodeling are synthesized in an inactive conformation, then activated by proteolytic cleavage Zymogen Pepsinogen Chymotrypsinogen Trypsinogen Procarboxypeptidase Proelastase Prothrombin Fibrinogen Factor VII Factor X Proinsulin Procollagen Procollagenase Active Enzyme Pepsin Chymotrypsin Trypsin Carboxypeptidase Elastase Thrombin Fibrin Factor VIIa Factor Xa Insulin Collagen Collagenase Function protein digestion blood clot formation plasma glucose homeostasis component of skin and bone remodeling processes during metamorphosis, etc.

Physiological significance of zymogen activation
Prevent autodigestion of the secretory organ itself. For example, premature activation of pancreatic zymogens ( trypsinogen, chymotrypsinogen and proelastase) leads to the condition of acute pancreatitis Keep a rapid and amplified response to condition changes because zymogens are storage of enzymes.

Isozymes Isoenzymes: Multiple forms of an enzyme which catalyze the same reaction, but differ from each other in their amino sequence, physicochemical properties and immuno characters. Isoenzymes maybe encoded by different genes or alleles. Isoenzymes may also come from translation of different mRNAs which are transcripted from the same gene.

What is an isozyme? (1) Isozymes are physically distinct forms of the same enzyme. (2) Isozymes may differ from each other by differences in their amino acid sequences or by the presence of different posttranslational modifications in each isozyme. (3) The relative abundance of different isozymes varies for different tissues. The ability to control which isozymes are expressed in a particular cell allows each cell to adjust the enzyme activity based on the specific conditions that exist in the cell.

Lactate dehydrogenase (LDH)
Isoenzymes Lactate dehydrogenase (LDH) LDH is a tetramer of two non-identical subunits H subunit M subunit Tetramer LDH (M.W. 130,000) Subunit: H (Chr12) M (Chr11) Each monomer can be either heart or muscle type Five different isozymes of lactate dehydrogenase exist: H4, H3M, H2M2, HM3, and M4

Tissue distribution specificity
Isozymes are enzymes with slightly different subunits Quaternary forms of Lactate Dehydrogenase (LDH) LDH

All of the lactate dehydrogenase isozymes catalyze the interconversion between lactate and pyruvate

Each isozyme of lactate dehydrogenase can be separated by chromatography
+ - Different tissues have different levels of each of the lactate dehydrogenase isozymes During myocardial infarction, endothelial cells rupture, releasing their contents into the bloodstream. This increases the serum levels for lactate dehydrogenase isozymes 1 and 2 and can be used as a diagnostic tool.

For each lane, the five different isozymes of lactate dehydrogenase are number 1-5 (with the isozyme containing all heart type monomers being 1 and all muscle type monomers being 5). The lane labeled “HEART” is from the serum of a patient with a myocardial infarction (heart attack), the lane labeled “NORMAL” is normal serum, and the lane labeled “LIVER” is from a patient with liver disease.

Myocardial infarction
Diagnostic Enzyme Analysis Normal serum Myocardial infarction Acute hepatitis

Regulation of enzyme quantity
（1）synthesis induction repression （2）degradation Cells can synthesize specific enzymes in response to changing metabolic needs, a process referred to as enzyme induction. The synthesis of certain enzymes may also be specifically inhibited, a process referred to as enzyme repression.

Drug design: Transition state analogues
Enzymes have evolved to recognize the transition state of the reaction they catalyze To design an enzyme inhibitor, we should try to mimic the transition state of the reaction, not the substrates or products

Clinical applications
Fundamental concepts: Plasma specific or plasma functional enzymes: normally present in the plasma and have specific functions. High activities in plasma than in the tissues. Synthesized in liver and enter the circulation. Impairment in liver function or genetic disorder leads to a fall in the activities. Non-plasma specific or plasma non-functional enzymes: either totally absent or at a low concentration in plasma.

In the normal turnover of cells, intracellular enzymes are released into blood stream.
An organ damaged by diseases may elevate those enzymes.

Diagnostic applications
Usefulness: -enzyme assays provide important information concerning the presence and severity of diseases. -provide a means of monitoring the patient’s response Approaches; -measuring the enzymatic activities directly -used as agents to monitor the presence of substrates.

Therapeutic applications
Successful therapeutic uses - streptokinase: treating myocardial infarction; preventing the heart damage once administrated immediately after heart attack -asparaginase: tumor regression Several limits -can be rapidly inactivated or digested -may provoke allergic effects

summary Enzymes produced by living cells are specific biocatalysts which have high specificity and efficiency in catalytic reactions. Simple enzymes and conjugated enzymes cofactors: Coenzymes prosthetic groups Active site of enzyme. Essential groups

They includes T, pH, [S], [E], activator, and the inhibitors.
Enzyme kinetics studies on the factors which affect the velocity of enzyme reaction. They includes T, pH, [S], [E], activator, and the inhibitors. Michaelis-Menten equation. Lineweaver-Burk double-reciprocal plot Km equals to the substrate concentration at V0 =Vmax/2.

In competitive inhibition reactions, the apparent Km is increased, while Vmax is not affected.
In noncompetitive inhibition reaction, Km is not affected, while Vmax is lowered. In uncompetitive inhibition reaction, Km and Vmax are all lowered. 1/[S] 1/v 1/vmax -1/Km Slope=Km/vmax +inhibitor -1/(Km(1+[I]/KI)) +inhibitor 1/[S] 1/v 1/vmax -1/Km Slope=Km/vmax (1+[I]/KI)/Vmax Slope= Km(1+[I]/KI)/vmax +inhibitor 1/[S] 1/v 1/vmax -1/Km Slope=Km/vmax (1+ KI/[I])/Vmax Slope= Km/vmax

Exploring the old and deducing the new makes a teacher.
Enzyme regulation: Allosteric regulation; Zymogen; Isozyme; Covalent modification; Exploring the old and deducing the new makes a teacher.

Thank you!

Chapter 3 Enzyme1 introduction to enzymes

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Chapter 3 Enzyme1 introduction to enzymes

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