CHEM 4311 General Biochemistry I Fall 2012

Chapter 13-15 Enzyme EXAM - October 19

Instructor: Office: Phone: email: Office hours Dr. Khairul I Ansari 316CPB 817-272-0616 kansari@uta.edu 12 am 1:30 pm Tuesday &.Thursday

Chapter 13 Enzyme Kinetics

Virtually All Reactions in Cells Are Mediated by Enzymes

Enzymes catalyze thermodynamically favorable reactions, causing them to proceed at extraordinarily rapid rates Enzymes provide cells with the ability to exert kinetic control over thermodynamic potentiality Living systems use enzymes to accelerate and control the rates of vitally important biochemical reactions Enzymes are the agents of metabolic function

Virtually All Reactions in Cells Are Mediated by Enzymes

C6H12O6 + 6O2 6CO2 + 6H20 + 2870kJ Energy

Figure 13.1 Reaction profile showing the large free energy of activation for glucose oxidation. Enzymes lower G, thereby accelerating rate.

13.1 What Characteristic Features Define Enzymes?

Catalytic power is defined as the ratio of the enzyme-catalyzed rate of a reaction to the uncatalyzed rate Specificity is the term used to define the selectivity of enzymes for their substrates Regulation of enzyme activity ensures that the rate of metabolic reactions is appropriate to cellular requirements Enzyme nomenclature provides a systematic way of naming metabolic reactions Coenzymes and cofactors are nonprotein components essential to enzyme activity.

13.1 What Characteristic Features Define Enzymes?

Enzymes can accelerate reactions as much as 1021 over uncatalyzed rates Urease is a good example: Catalyzed rate: 3x104/sec Uncatalyzed rate: 3x10 -10/sec Ratio (catalytic power) is 1x1014

Specificity
Enzymes selectively recognize proper substrates over other molecules Enzymes produce products in very high yields often much greater than 95% Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product thats formed.

Enzyme Nomenclature Provides a Systematic Way of Naming Metabolic Reactions

Coenzymes and Cofactors Are Nonprotein Components Essential to Enzyme Activity

WHAT IS a -Cofactor? -Coenzyme? -Prosthetic group? -Apoenzyme? -Holoenzyme?

13.2 Can the Rate of an Enzyme-Catalyzed Reaction Be Defined in a Mathematical Way?

Kinetics is the branch of science concerned with the rates of reactions Enzyme kinetics seeks to determine the maximum reaction velocity that enzymes can attain and the binding affinities for substrates and inhibitors Analysis of enzyme rates yields insights into enzyme mechanisms and metabolic pathways This information can be exploited to control and manipulate the course of metabolic events

Several Kinetics Terms to Understand rate or velocity rate constant rate law order of a reaction molecularity of a reaction

Chemical Kinetics Provides a Foundation for Exploring Enzyme Kinetics

Consider a reaction of overall stoichiometry as shown:

A P

d[P] d[ A] = dt dt [ A] v= = k[ A] dt v=
The rate is proportional to the concentration of A

Chemical Kinetics Provides a Foundation for Exploring Enzyme Kinetics The simple elementary reac on of A P is a firstorder reaction This is a unimolecular reaction For a bimolecular reaction, the rate law is: v = k[A][B] Kinetics cannot prove a reaction mechanism Kinetics can only rule out various alternative hypotheses because they dont fit the data

The Time-Course of a First-Order Reaction

Figure 13.4 Plot of the course of a first-order reaction. The half-time, t1/2 is the time for one-half of the starting amount of A to disappear.

Catalysts Lower the Free Energy of Activation for a Reaction A typical enzyme-catalyzed reaction must pass through a transition state The transition state sits at the apex of the energy profile in the energy diagram The reaction rate is proportional to the concentration of reactant molecules with the transition-state energy This energy barrier is known as the free energy of activation Decreasing G increases the reaction rate The activation energy is related to the rate constant by:

k = Ae G/ RT

Catalysts Lower the Free Energy of Activation for a Reaction

Figure 13.5 Energy diagram for a chemical reaction (AP) and the effects of (a) raising the temperature from T1 to T2, or (b) adding a catalyst.

The Transition State Understand the difference between G and G The overall free energy change for a reaction, G, is
related to the equilibrium constant The free energy of activation for a reaction, G, is related to the rate constant It is extremely important to appreciate this distinction

13.3 What Equations Define the Kinetics of EnzymeCatalyzed Reactions?

Simple first-order reactions display a plot of the reaction rate as a function of reactant concentration that is a straight line (Figure 13.6) Enzyme-catalyzed reactions are more complicated At low concentrations of the enzyme substrate, the rate is proportional to S, as in a first-order reaction At higher concentrations of substrate, the enzyme reaction approaches zero-order kinetics This behavior is a saturation effect

13.3 What Equations Define the Kinetics of EnzymeCatalyzed Reactions?

Figure 13.6 A plot of versus [A] for the unimolecular chemical reaction, AP, yields a straight line having a slope equal to k. This reaction is a firstorder reaction.

As [S] increases, kinetic behavior changes from 1st order to zero-order kinetics

Figure 13.7 Substrate saturation curve for an enzyme-catalyzed reaction.

The Michaelis-Menten Equation is the Fundamental Equation of Enzyme Kinetics Louis Michaelis and Maud Menten's theory assumes the formation of an enzyme-substrate complex assumes that the ES complex is in rapid equilibrium with free enzyme assumes that the breakdown of ES to form products is slower than 1) formation of ES and 2) breakdown of ES to re-form E and S

[ES] Remains Constant Through Much of the Enzyme Reaction Time Course in Michaelis-Menten Kinetics

Figure 13.8 Time course for a typical enzyme-catalyzed reaction obeying the Michaelis-Menten, Briggs-Haldane models for enzyme kinetics. The early state of the time course is shown in greater magnification in the bottom graph.

The Michaelis-Menten equation

v= Vmax [S] K m + [S]
Vmax = k2 [E T ]

where

and

Km

( k1 + k2 ) =
k1

Understanding Km
The "kinetic activator constant" Km is a constant 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 Small Km means tight binding; high Km means weak binding

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 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

The Turnover Number Defines the Activity of One Enzyme Molecule 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

The Turnover Number Defines the Activity of One Enzyme Molecule

Turn over number (Kca.s-1) of Catalase DNA Plymerase I 40,000,000 15

The Ratio kcat/Km Defines the Catalytic Efficiency of an Enzyme

The catalytic efficiency: kcat/Km An estimate of "how perfect" the enzyme is kcat/Km is an apparent second-order rate constant It measures how well the enzyme performs when S is low The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together

Enzymatic Activity is Strongly Influenced by pH Enzyme-substrate recognition and catalysis are greatly dependent on pH Enzymes have a variety of ionizable side chains that determine its secondary and tertiary structure and also affect events in the active site The substrate may also have ionizable groups Enzymes are usually active only over a limited range of pH The effects of pH may be due to effects on Km or Vmax or both

Enzymatic Activity is Strongly Influenced by pH

Figure 13.11 The pH activity profiles of four different enzymes.

The Response of Enzymatic Activity to Temperature is Complex Rates of enzyme-catalyzed reactions generally increase with increasing temperature However, at temperatures above 50to 60C, enzymes typically show a decline in activity Two effects here: Enzyme rate typically doubles in rate for ever 10 C as long as the enzyme is stable and active At higher temperatures, the protein becomes unstable and denaturation occurs

The Response of Enzymatic Activity to Temperature is Complex

Figure 13.12 The effect of temperature on enzyme activity.

13.4 What Can Be Learned from the Inhibition of Enzyme Activity?

Enzymes may be inhibited reversibly or irreversibly Reversible inhibitors may bind at the active site or at some other site Enzymes may also be inhibited in an irreversible manner Penicillin is an irreversible suicide inhibitor

Cyanide is an effective reversible inhibitor of cytochrome oxidase

Reversible Inhibitors May Bind at the Active Site or at Some Other Site
Competitive Inhibitors Compete With Substrate for the Same Site on the Enzyme
Methanol & Ethanol are competitor of Lliver alcohol dehydrogenase (ADH)

Pure Noncompetitive Inhibition where S and I bind to different sites on the enzyme Mixed Noncompetitive Inhibition: binding of I by E influences binding of S by E Uncompetitive Inhibition, where I combines only with E, but not with ES

Enzymes Can Be Inhibited Irreversibly

Figure 13.18 Penicillin is an irreversible inhibitor of the enzyme glycoprotein peptidease, which catalyzes an essential step in bacterial cell all synthesis.

13.5 - What Is the Kinetic Behavior of Enzymes Catalyzing Bimolecular Reactions? Enzymes often catalyze reactions involving two (or more) substrates Bisubstrate reactions may be sequential or singledisplacement reactions or double-displacement (pingpong) reactions Single-displacement reactions can be of two distinct classes: 1. Random, where either substrate may bind first, followed by the other substrate 2. Ordered, where a leading substrate binds first, followed by the other substrate

Conversion of AEB to PEQ is the Rate-Limiting Step in Random, Single-Displacement Reactions

In this type of sequential reaction, all possible binary enzyme-substrate and enzyme-product complexes are formed rapidly and reversibly when enzyme is added to a reaction mixture containing A, B, P, and Q.

In an Ordered, Single-Displacement Reaction, the Leading Substrate Must Bind First

The leading substrate (A) binds first, followed by B. Reaction between A and B occurs in the ternary complex and is usually followed by an ordered release of the products, P and Q.

An Alternative way of Portraying the Ordered, SingleDisplacement Reaction

The Double Displacement (Ping-Pong) Reaction

Double-Displacement (Ping-Pong) reactions proceed via formation of a covalently modified enzyme intermediate. Reactions conforming to this kinetic pattern are characterized by the fact that the product of the enzymes reaction with A (called P in the above scheme) is released prior to reaction of the enzyme with the second substrate, B.

An Alternative Presentation of the DoubleDisplacement (Ping-Pong) Reaction

Glutamate:aspartate Aminotransferase

Figure 13.23 An enzyme conforming to a doubledisplacement bisubstrate mechanism.

13.7 How Can Enzymes Be So Specific?

The Lock and key hypothesis was the first explanation for specificity The Induced fit hypothesis provides a more accurate description of specificity Induced fit favors formation of the transition state Specificity and reactivity are often linked. In the hexokinase reaction, binding of glucose in the active site induces a conformational change in the enzyme that causes the two domains of hexokinase to close around the substrate, creating the catalytic site

13.7 How Can Enzymes Be So Specific?

Figure 13.24 A drawing, roughly to scale, of H2O, glycerol, glucose, and an idealized hexokinase molecule. Binding of glucose in the active site induces a conformational change in the enzyme that causes the two domains of hexokinase to close around the substrate, creating the catalytic site.

13.7 Are All Enzymes Proteins?

Ribozymes - segments of RNA that display enzyme activity in the absence of protein Examples: RNase P and peptidyl transferase Abzymes - antibodies raised to bind the transition state of a reaction of interest

Antibody Molecules Can Have Catalytic Activity

Multiple sclerosis MS basic protein is proteolyses by antibody Degradation of factor VIII in hemophilia

13.8 Is It Possible to Design An Enzyme to Catalyze Any Desired Reaction?

A known enzyme can be engineered by in vitro mutagenesis, replacing active site residues with new ones that might catalyze a desired reaction Another approach attempts to design a totally new protein with the desired structure and activity This latter approach often begins with studies in silico i.e., computer modeling Protein folding and stability issues make this approach more difficult Further, the cellular environment may provide complications not apparent in the computer modeling

Chapter 14 Mechanisms of Enzyme Action

14.1 What Are the Magnitudes of Enzyme-Induced Rate Accelerations? Enzymes are powerful catalysts The large rate accelerations of enzymes (107 to 1015) correspond to large changes in the free energy of activation for the reaction All reactions pass through a transition state on the reaction pathway The active sites of enzymes bind the transition state of the reaction more tightly than the substrate By doing so, enzymes stabilize the transition state and lower the activation energy of the reaction

14.2 What Role Does Transition-State Stabilization Play in Enzyme Catalysis?

The catalytic role of an enzyme is to reduce the energy barrier between substrate S and transition state X Rate acceleration by an enzyme means that the energy barrier between ES and EX must be smaller than the barrier between S and X This means that the enzyme must stabilize the EX transition state more than it stabilizes ES

14.3 How Does Destabilization of ES Affect Enzyme Catalysis? Raising the energy of ES raises the rate For a given energy of EX, raising the energy of ES will increase the catalyzed rate This is accomplished by a) loss of entropy due to formation of ES b) destabilization of ES by strain distortion desolvation

14.3 How Does Destabilization of ES Affect Enzyme Catalysis?

Figure 14.2 The intrinsic binding energy of ES is compensated by entropy loss due to binding of E and S and by destabilization due to strain and distortion.

14.3 How Does Destabilization of ES Affect Enzyme Catalysis?

Figure 14.3 (a) Catalysis does not occur if ES and X are equally stabilized. (b) Catalysis will occur if X is stabilized more than ES.

14.3 How Does Destabilization of ES Affect Enzyme Catalysis?

Figure 14.4 (a) Formation of the ES complex results in entropy loss. The ES complex is a more highly ordered, low-entropy state for the substrate.

14.3 How Does Destabilization of ES Affect Enzyme Catalysis?

Figure 14.4 (b) Substrates typically lose waters of hydration in the formation in the formation of the ES complex. Desolvation raises the energy of the ES complex, making it more reactive.

14.3 How Does Destabilization of ES Affect Enzyme Catalysis?

Figure 14.4 (c) Electrostatic destabilization of a substrate may arise from juxtaposition of like charges in the active site. If charge repulsion is relieved in the reaction, electrostatic destabilization can result in a rate increase.

14.4 How Tightly Do Transition-State Analogs Bind to the Active Site?

Very tight binding to the active site The affinity of the enzyme for the transition state may be 10 -20 to 10-26 M! Transition state exists only for 10-14 to 10-13 sec. this time is equivalent to a vibration of a bond. Transition state analogs (TSAs) are stable molecules that are chemically and structurally similar to the transition state Proline racemase was the first case

Transition-State Analogs Make Our World Better

Enzymes are often targets for drugs and other beneficial agents Transition-state analogs often make ideal enzyme inhibitors Enalapril and Aliskiren lower blood pressure Statins lower serum cholesterol Protease inhibitors are AIDS drugs Juvenile hormone esterase is a pesticide target Tamiflu is a viral neuraminidase inhibitor

Transition-State Analogs Make Our World Better

Statins such as Lipitor are powerful cholesterol-lowering drugs, because they are transition-state analog inhibitors of HMG-CoA (3-hydroxy-3methylglutaryl-coenzyme A ) reductase, a key enzyme in the biosynthetic pathway for cholesterol.

Transition-State Analogs Make Our World Better

One strategy for controlling insect populations is to alter the actions of juvenile hormone, a terpene-based substance that regulates insect life cycle processes. Levels of juvenile hormone are controlled by juvenile hormone esterase (JHE), and inhibition of JHE is toxic to insects. OTEP (figure) is a potent transition state-analog inhibitor of JHE.

Tamiflu is a Viral Neuraminidase Inhibitor

Influenza is a serious illness that affects 5% to 15% of the earths population each year and results in up to 500,000 deaths annually. Neuraminidase is a major glycoprotein on the influenza virus membrane envelope that is essential for viral replication and infectivity. Tamiflu is a neuraminidase inhibitor and antiviral agent based on the transition state of the neuraminidase reaction.

How many other drug targets might there be?

The human genome contains approximately 20,000 genes How many might be targets for drug therapy? More than 3000 experimental drugs are presently under study and testing These and many future drugs will be designed as transition-state analog inhibitors

Reaction mechanisms How to read and write reaction mechanisms

The custom of writing chemical reaction mechanisms with electron dots and curved arrows began with Gilbert Newton Lewis and Sir Robert Robinson And with a review of the concepts of valence electrons and formal charge Formal charge = group number nonbonding electrons ( shared electrons) Electronegativity is also important: F>O>N>C>H
For example, in C-N bond, the N should be viewed as more electron rich and carbon as electron deficient

How to read and write mechanisms

In written mechanisms, a curved arrow shows the movement of an electron pair And thus the movement of a pair of electrons from a filled orbital to an empty one A full arrowhead represents an electron pair A half arrowhead represents a single electron For a bond-breaking event, the arrow begins in the middle of the bond For example, bond-breaking event bond-making event

How to read and write mechanisms

It has been estimated that 75% of the steps in enzyme reaction mechanisms are proton (H+) transfers. If the proton is donated or accepted by a group on the enzyme, it is often convenient (and traditional) to represent the group as B, for base, even if B is protonated and behaving as an acid:

How to read and write mechanisms

It is important to appreciate that a proton transfer can change a nucleophile (a species that donates an electronpair) into an electrophile (a species that accept an electronpair), and vice versa. Thus, it is necessary to consider: The protonation states of substrate and active-site residues How pKa values can change in the environment of the active site For example, an active-site histidine, which might normally be protonated, can be deprotonated by another group and then act as a base, accepting a proton from the substrate

How to read and write mechanisms

Water can often act as an acid or base at the active site through proton transfer with an assisting active-site residue:

This type of chemistry is the basis for general acid-base catalysis (discussed on pages 430-431).

14.5 What Are the Mechanisms of Catalysis?

Protein motions are essential to enzyme catalysis Enzymes facilitate formation of near-attack complexes (NAC) Enzyme reaction mechanism involves 1. Covalent bond formation (Covalent catalysis) 2. General acid-base catalysis 3. Metal ion catalysis 4. Proximity and favorable orientation of reactant (Low-barrier hydrogen bonds)

Enzymes facilitate formation of near-attack complexes

X-ray crystal structure studies and computer modeling have shown that the reacting atoms and catalytic groups are precisely positioned for their roles Such preorganization selects substrate conformations in which the reacting atoms are in van der Waals contact and at an angle resembling the bond to be formed in the transition state NACs are precursors to reaction transition states

Enzymes facilitate formation of near-attack complexes

NACs are precursors to transition states In the absence of an enzyme, potential reactant molecules adopt a NAC only about 0.0001% of the time On the other hand, NACs have been shown to form in enzyme active sites from 1% to 70% of the time
Figure 14.7 In an enzyme active site, the NAC forms more readily than in the uncatalyzed reaction. The energy separation between the NAC and the transition state is approximately the same in the presence and absence of the enzyme.

Protein Motions Are Essential to Enzyme Catalysis

Proteins are constantly moving bonds vibrate, side chains bend and rotate, backbone loops wiggle and sway, and whole domains move as a unit Enzymes depend on such motions to provoke and direct catalytic events Protein motions support catalysis in several ways. Active site conformation changes can: 1. 2. 3. 4. 5. Assist substrate binding Bring catalytic groups into position Induce formation of NACs Assist in bond making and bond breaking Facilitate conversion of substrate to product

Protein Motions Are Essential to Enzyme Catalysis

Figure 14.10 Catalysis in enyzme active sites depends on motion of active-site residues. Several active-site residues undergo greater motion during catalysis than residues elsewhere in the protein.

Mechanisms of Catalysis 1. Covalent Catalysis

Some enzymes derive much of their rate acceleration from formation of covalent bonds between enzyme and substrate The side chains of amino acids in proteins offer a variety of nucleophilic centers for catalysis These groups readily attack electrophilic centers of substrates, forming covalent enzyme-substrate complexes The covalent intermediate can be attacked in a second step by water or by a second substrate, forming the desired product

Covalent Catalysis

Figure 14.11 Examples of covalent bond formation between enzyme and substrate. A nucleophilic center X: attacks the anomeric carbon of a glycoside, forming a glucosyl enzyme intermediate.

Covalent Catalysis

Mechanisms of Catalysis 2. Acid-Base Catalysis

Catalysis in which a proton is transferred in the transition state "Specific" acid-base catalysis involves H+ or OH- that diffuses into the catalytic center "General" acid-base catalysis involves acids and bases other than H+ and OH These other acids and bases facilitate transfer of H+ in the transition state

Mechanisms of Catalysis 3. Low-Barrier Hydrogen Bonds (LBHBs)

A weak hydrogen bond may become an LBHB in a transient intermediate The energy released in forming the LBHB is used to help the reaction by lowering the activation barrier

Mechanisms of Catalysis 4. Quantum Mechanical Tunneling

Tunneling provides a path around the usual energy of activation for steps in chemical reactions Many enzymes exploit this According to quantum theory, there is a finite probability that any particle can appear on the other side of an activation barrier for a reaction step The likelihood of tunneling depends on the distance over which a particle must move Only protons and electrons have a significant probability of tunneling

Tunneling between donor and acceptor

Figure 14.13d If the distance for particle transfer is sufficiently small, overlap of probability functions (red) permit efficient quantum mechanical tunneling between donor (D) and acceptor (A)

Mechanisms of Catalysis 5. Metal Ion Catalysis

Reaction by Metalloenzyme Eg Thermolysin and Zn

Figure 14.14 Thermolysin is an endoprotease with a catalytic Zn2+ ion in the active site. The Zn2+ ion stabilizes the buildup of negative charge on the peptide carbonyl oxygen, as a glutamate residue deprotonates water, promoting hydroxide attack on the carbonyl carbon.

How Do Active-Site Residues Interact to Support Catalysis?

About half of the amino acids engage directly in catalytic effects in enzyme active sites Other residues may function in secondary roles in the active site: Raising or lowering catalytic residue pKa values Orientation of catalytic residues Charge stabilization Proton transfers via hydrogen tunneling

Typical Enzyme Mechanisms?

Enzyme and substrate become linked in a covalent bond at one or more points in the reaction pathway The formation of the covalent bond provides chemistry that speeds the reaction Serine proteases Serine in active site of Serine proteases- act as nucleophilic amino acid and facilitate cleavage of peptide bonds of the substrate. The active site of Serine proteases- contain Asp120, His57 and Ser 195 position. Asp102 functions only to orient His57 His57 acts as a general acid and base Ser195 forms a covalent bond with peptide to be cleaved Covalent bond formation turns a trigonal C into a tetrahedral C The tetrahedral oxyanion intermediate is stabilized by the backbone N-H groups of Gly193 and Ser195

Aspartic Proteases
Pepsin, chymosin, cathepsin D, renin and HIV-1 protease All involve two Asp residues at the active site These two Asp residues work together as general acid-base catalysts Most aspartic proteases have a tertiary structure consisting of two lobes (N-terminal and C-terminal) with approximate two-fold symmetry HIV-1 protease is a homodimer

 Aspartic proteases show one relatively low pKa, and one relatively high pKa This was once thought to represent pKa values of the two aspartate residues, but this is no longer believed to be the case Instead, molecular dynamics simulations show that aspartic proteases employ low-barrier hydrogen bonds (LBHBs) in their mechanism The predominant catalytic factor in aspartic proteases is general acid-base catalysis

Protease inhibitors as AIDS drugs If HIV-1 protease can be selectively inhibited, then new HIV particles cannot form Several novel protease inhibitors are currently marketed as AIDS drugs Many such inhibitors work in a culture dish However, a successful drug must be able to kill the virus in a human subject without blocking other essential proteases in the body

Chapter 15 Enzyme Regulation

15.1 What Factors Influence Enzymatic Activity?

1. The availability of substrates and cofactors usually determines how fast the reaction goes 2. As product accumulates, the apparent rate of the enzymatic reaction will decrease 3. Genetic regulation of enzyme synthesis ( by induction or repression) and decay determines the amount of enzyme present at any moment 4. Enzyme activity can be regulated allosterically 5. Enzyme activity can be regulated through covalent modification (for example phosphorylation of kinases) 6. Zymogens, isozymes, and modulator proteins may play a role

Two prominent way to regulate enzyme

1. By increasing or decreasing enzyme molecules-by regulating gene expression (induction or suppression) and protein degradation 2. Increasing or decreasing intrinsic activity of each enzyme molecules-principally by allosteric regulation or covalent modifications

Allosteric Regulation?
In allosteric regulation (feedback control) the effector molecule binds at the protein's allosteric site (that is, a site other than the protein's active site). If the effectors that enhance the activity - allosteric activators If the effectors that decrease the activity -allosteric inhibitors. Enzymes situated at key steps in metabolic pathways are modulated by allosteric effectors These effectors are usually produced elsewhere in the pathway Features of allosteric regulation 1. Does not follow Michaelis-Menten equation 2. Feedback inhibition is different than normal enzyme inhibitor 3. The effector molecules may activate the allosteric enzyme or stimulate its activity 4. Most allosteric enzymes have oligomeraic organization 5. The interaction of the enzyme with effector molecule causes conformation changes in the active site of the enzyme.

15.3 Can Allosteric Regulation by Conformational

- Allosteric proteins can exist in two states: R (relaxed) and T (taut) - In this model, all the subunits of an oligomer must be in the same state - T state predominates in the absence of substrate S - S binds much tighter to R than to T

Figure 15.7 Allosteric effects: A and I binding to R and T, respectively.

The Sequential Model for Allosteric Regulation is Based on Ligand-Induced Conformation Changes
An alternative model proposed by Koshland, Nemethy, and Filmer (the KNF model) relies on the idea that ligand binding triggers a conformation change in a protein If the protein is oligomeric, ligand-induced conformation changes in one subunit may lead to conformation changes in adjacent subunits The KNF model explains how ligand-induced conformation changes could cause subunits to adopt conformations with little affinity for the ligand i.e., negative cooperativity The KNF model is termed the sequential model

Enzyme activity can be regulated by covalent Modification eg Phosphorylation, acetylation etc

The interconvertible enzymes can by reversibly modified Enzyme activity can be regulated through reversible phosphorylation This is the most prominent form of covalent modification in cellular regulation Phosphorylation is accomplished by protein kinases Each protein kinase targets specific proteins for phosphorylation Phosphoprotein phosphatases catalyze the reverse reaction removing phosphoryl groups from proteins Kinases and phosphatases themselves are targets of regulation

15.4 What Kinds of Covalent Modification Regulate the Activity of Enzymes?

Protein kinases phosphorylate Ser, Thr, and Tyr residues in target proteins Kinases typically recognize specific amino acid sequences in their targets In spite of this specificity, all kinases share a common catalytic mechanism based on a conserved core kinase domain of about 260 residues (see Figure 15.9) Kinases are often regulated by intrasteric control, in which a regulatory subunit (or domain) has a pseudosubstrate sequence that mimics the target sequence, minus the phosphorylatable residue

Enzyme activity can be regulated by covalent Modification

Figure 15.1 Enzyme regulation by reversible covalent modification.

Phosphorylation is Not the Only Form of Covalent Modification that Regulates Protein Function

Several hundred different chemical modifications of proteins have been discovered Only a few of these are used to achieve metabolic regulation through reversible conversion of an enzyme between active and inactive forms

Enzyme activity can be regulated by covalent Modification eg Phosphorylation, acetylation

Acetylation is a prominent modification for the regulation of metabolic enzymes Acetylation of an -NH3+ group on a Lys residue changes it from a positively charged amino group to a neutral amide This change may have consequences for protein structure and thus function The acetylating enzyme is termed an acetyl-CoA-dependent lysine acetyltransferase or KAT More than 30 KATs are known in mammals Deacetylation by KDACs (lysine deacetylases) reverse the effects of acetylation

In natural situation enzyme activity is also regulated by presence of zymogens, isozymes and modulator protein
Zymogens are inactive precursors of enzymes. Typically, proteolytic cleavage produces the active enzyme.

Figure 15.2 Proinsulin is an 86residue precursor to insulin

Figure 15.3 The proteolytic activation of chymotrypsinogen

Proteolytic Enzymes of the Digestive Tract

Acetylation in Enzyme Regulation

Proteomics studies show that acetylation of metabolic enzymes is an important mechanism for regulating the flow of metabolic substrates (carbohydrates and fats, for example) through the central metabolic pathways Acetylation activates some enzymes and inhibits others Cellular levels of major metabolic fuels such as glucose, fatty acids, and amino acids influence the degree of acetylation The KDACs include sirtuins, a class of NAD+-dependent protein deacetylating enzymes Sirtuins are implicated in energy metabolism and longevity

Acetylation in Enzyme Regulation

Figure 15.11 Activation of malate dehydrogenase by acetylation

Isozymes Are Enzymes With Slightly Different Subunits

Figure 15.5 The isozymes of lactate dehydrogenase (LDH).

15.5 Are Some Enzymes Controlled by Both Allosteric Regulation and Covalent Modification? Glycogen phosphorylase (GP) is an example of the many enzymes that are regulated both by allosteric controls and by covalent modification GP cleaves glucose units from nonreducing ends of glycogen This converts glycogen into readily usable fuel in the form of glucose-1-phosphate This is a phosphorolysis reaction Muscle GP is a dimer of identical subunits, each with PLP covalently linked There is an allosteric effector site at the subunit interface

GP converts glycogen into readily usable fuel in the form of glucose-1-phosphate

Figure 15.12 The glycogen phosphorylase reaction

Phosphoglucomutase converts glucose-1-P into the glycolytic substrate, glucose-6-P

Figure 15.13 The phosphoglucomutase reaction.

Glycogen Phosphorylase Activity is Regulated Allosterically Muscle glycogen phosphorylase shows cooperativity in substrate binding ATP and glucose-6-P are allosteric inhibitors of glycogen phosphorylase AMP is an allosteric activator of glycogen phosphorylase When ATP and glucose-6-P are abundant, glycogen breakdown is inhibited When cellular energy reserves are low (i.e., high [AMP] and low [ATP] and [G-6-P]) glycogen catabolism is stimulated

Glycogen Phosphorylase Activity is Regulated Allosterically

Figure 15.15 v versus S curves for glycogen phosphorylase. (a)The response to the concentration of the substrate phosphate (Pi). (b)ATP is a feedback inhibitor. (c)AMP is a positive effector. It binds at the same site as ATP.

Glycogen phosphorylase conforms to the MWC model

The active form of the enzyme is designated the R state The inactive form of the enzyme is denoted the T state AMP promotes the conversion to the active state ATP, glucose-6-P, and caffeine favor conversion to the inactive T state A significant conformation change occurs at the subunit interface between the T and R state This conformational change at the interface is linked to a structural change at the active site that affects catalysis

Hemoglobin
A classic example of allostery Hemoglobin and myoglobin are oxygen- transport and oxygenstorage proteins, respectively Compare the oxygen-binding curves for hemoglobin and myoglobin Myoglobin is monomeric; hemoglobin is tetrameric Mb: 153 aa, 17,200 MW Hb: two chains of 141 residues, 2 chains of 146 residues

Figure 15.21 O2-binding curves for hemoglobin and myoglobin

Mb and Hb use heme to bind Fe2+

Figure 15.23 Heme is formed when protoporphyrin IX binds Fe2+

Fe2+ is coordinated by His F8

Iron interacts with six ligands in Hb and Mb Four of these are the N atoms of the porphyrin A fifth ligand is donated by the imidazole side chain of amino acid residue His F8 (This residue is on the sixth or F helix, and it is the 8th residue in the helix, thus the name.) When Mb or Hb bind oxygen, the O2 molecule adds to the heme iron as the sixth ligand The O2 molecule is tilted relative to a perpendicular to the heme plane

Myoglobin Structure

Mb is a monomeric heme protein Mb polypeptide "cradles" the heme group Fe in Mb is Fe2+ - ferrous iron - the form that binds oxygen Oxidation of Fe yields 3+ charge - ferric iron Mb with Fe3+ is called metmyoglobin and does not bind oxygen

O2 Binding Alters Mb Conformation

In deoxymyoglobin, the ferrous ion actually lies 0.055 nm above the plane of the heme When oxygen binds to Fe in heme of Mb, the heme Fe is drawn toward the plane of the porphyrin ring With oxygen bound, the Fe2+ atom is only 0.026 nm above the plane For Mb, this small change has little consequence But a similar change in Hb initiates a series of conformational changes that are transmitted to adjacent subunits

Cooperative Binding of Oxygen Influences Hemoglobin Function

Mb, an oxygen-storage protein, has a greater affinity for oxygen at all oxygen pressures Hb is different it must bind oxygen in lungs and release it in capillaries Hb becomes saturated with O2 in the lungs, where the partial pressure of O2 is about 100 torr In capillaries, pO2 is about 40 torr, and oxygen is released from Hb The binding of O2 to Hb is cooperative binding of oxygen to the first subunit makes binding to the other subunits more favorable

The Conformation Change

The secret of Mb and Hb Oxygen binding changes the Mb conformation Without oxygen bound, Fe2+ is out of heme plane Oxygen binding pulls the Fe2+ into the heme plane Fe2+ pulls its His F8 ligand along with it The F helix moves when oxygen binds Total movement of Fe2+ is 0.029 nm i.e., 0.29 This change means little to Mb, but lots to Hb!

Oxygen Binding by Hb Induces a Quaternary Structure Change

When deoxy-Hb crystals are exposed to oxygen, they shatter. Evidence of a large-scale structural change One alpha-beta pair moves relative to the other by 15 degrees upon oxygen binding This massive change is induced by movement of Fe by 0.039 nm when oxygen binds

Fe2+ Movement by Less Than 0.04 nm Induces the Conformation Change in Hb

In deoxy-Hb, the iron atom lies out of the heme plane by about 0.06 nm Upon O2 binding, the Fe2+ atom moves about 0.039 nm closer to the plane of the heme It is as if the O2 is drawing the heme iron into the plane This may seem like a trivial change, but its biological consequences are far-reaching As Fe2+ moves, it drags His F8 and the F helix with it This change is transmitted to the subunit interfaces, where conformation changes lead to the rupture of salt bridges

Fe2+ Movement by Less Than 0.04 nm Induces the Conformation Change in Hb

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