Chem 4311- Chapter13-15 Enzyme-10-12-12

Chapter 13-15 Enzyme

EXAM - October 19

Instructor: Dr. Khairul I Ansari

Office: 316CPB

Phone: 817-272-0616

email: [email protected]

Office hours 12 am – 1:30 pm Tuesday &.Thursday

CHEM 4311

General Biochemistry I

Fall 2012

Chapter 13Enzyme 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

Figure 13.1 Reaction profile showing the large free energy of activation for

glucose oxidation. Enzymes lower ∆G‡, thereby accelerating rate.

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

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 that’s 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:

• The rate is proportional to the concentration of A

A → P

v =d[P]

dt=

−d[A]

dt

v =−[A]

dt= k[A]

Chemical Kinetics Provides a Foundation for Exploring

Enzyme Kinetics

• The simple elementary reac5on of A→P is a first-order 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 don’t 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 (A→P)

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 Enzyme-

Catalyzed 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 Enzyme-

Catalyzed Reactions?

Figure 13.6 A plot of υ versus [A] for the unimolecular chemical reaction,

A→P, yields a straight line having a slope equal to k. This reaction is a first-

order 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 =V

max[S]

Km

+ [S]

whereV

max= k

2[E

T]

Km

=k

−1+ k

2( )k

1

and

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 40,000,000

DNA Plymerase I 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 50°to 60°C, enzymes typically show a decline in activity

• Two effects here:

– Enzyme rate typically doubles in rate for ever 10º 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

Competitive Inhibitors Compete With Substrate for the Same Site on the Enzyme

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

Reversible Inhibitors May Bind at the Active Site or at Some

Other Site

Methanol & Ethanol are competitor of

Lliver alcohol dehydrogenase (ADH)

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 single-

displacement reactions or double-displacement (ping-

pong) 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, Single-

Displacement 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 enzyme’s 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 Double-

Displacement (Ping-Pong) Reaction

Glutamate:aspartate Aminotransferase

Figure 13.23

An enzyme

conforming to a

double-

displacement

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 14Mechanisms 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


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.


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


Statins such as Lipitor are

powerful cholesterol-lowering

drugs, because they are

transition-state analog inhibitors

of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A )

reductase, a key enzyme in the

biosynthetic pathway for

cholesterol.


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 earth’s 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 – (½ shared electrons)

• Electronegativity is also important:

F > O > N > C > HFor 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


• 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:


• It is important to appreciate that a proton transfer can change a nucleophile (a species that donates an electron-pair) into an electrophile (a species that accept an electron-pair), 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


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. Assist substrate binding

2. Bring catalytic groups into position

3. Induce formation of NACs

4. Assist in bond making and bond breaking

5. 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


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


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


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)


5. Metal Ion Catalysis

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.

Reaction by Metalloenzyme

Eg Thermolysin and Zn

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 ProteasesPepsin, 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 regulation1. Does not follow Michaelis-Menten equation2. Feedback inhibition is different than normal enzyme inhibitor3. The effector molecules may activate the allosteric enzyme or

stimulate it’s activity4. Most allosteric enzymes have oligomeraic organization5. 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 phosphorylatableresidue

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 ε-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

Figure 15.2 Proinsulin is an 86-

residue precursor to insulin

Zymogens are inactive precursors

of enzymes. Typically, proteolytic

cleavage produces the active

enzyme.

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 oxygen-storage 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 α chains of 141 residues, 2 β 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 hemeiron as the sixth ligand

• The O2 molecule is tilted relative to a perpendicular to the hemeplane

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 Å

• 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

ERROR: stackunderflow

OFFENDING COMMAND: ~

STACK:

Documents

Chem 4311- Chapter13-15 Enzyme-10-12-12