1

1 Enzymes

It's true hard work never killed anybody, but I figure, why take the chance? Ronald Reagan

2

ENZYMES• Thermodynamic principles can be used to indicate

whether or not a reaction can take place spontaneously

• They do not, however, provide information about the rate at which a reaction will proceed

• Most biochemical reactions proceed so slowly at physiological temperatures that catalysis is essential for the reactions to proceed at a satisfactory rate in the cell

• At temperatures above absolute zero, all molecules possess vibrational energy which increases as the molecules are heated

• As the temperature rises, vibrating molecules are more likely to collide

• A chemical reaction occurs when colliding molecules possess a minimum amount of energy called the activation energy

• Not all collisions result in chemical reactions because only a fraction of the molecules have sufficient energy or the correct orientation to react

3

• Another way of increasing the likelihood of collisions, thereby the formation of product, is to increase the concentration of reactants

• In living systems, however, elevated temperatures may harm delicate biological structures and reactant concentrations are usually quite low

• The preferred catalysts in living systems, therefore, are enzymes, most of which are proteins (except for ribozymes which are RNA)

• Enzymes can increase the rate of a reaction by several orders of magnitude

• Enzymes do their job by decreasing activation energy, thereby increasing the percentage of substrate molecules that have sufficient energy to react

• Indeed, in the absence of enzymes, life as we know it would not be possible

4

• Enzymes do not affect ΔG (the position of the equilibrium) but speed up its attainment. The concentrations of substrate and product at equilibrium are not changed

The

Effec

t of

Cat

alys

is o

n

t

he A

ctiv

atio

n En

ergy

of

a Re

acti

on

5

The Basic Features of Enzymes1. Catalytic power• Enzymes accelerate reaction rates as much as 1016

over uncatalyzed levels, which is far greater than any synthetic catalysts can achieve• And enzymes accomplish these astounding feats in

dilute aqueous solutions under mild conditions of temperature and pH

2. Specificity• The action of enzymes is usually very specific. This

applies not only to the type of reaction being catalyzed (reaction specificity), but also to the nature of the substrates that are involved (substrate specificity)• Enzymes with low reaction specificity and low

substrate specificity are very rare• Intimate interaction between an enzyme and its

substrates occurs through molecular recognition based on structural complementarity

6

• Such mutual recognition is the basis for specificity. The specific site on the enzyme where substrate binds and catalysis occurs is called the active site

3. Regulation • Although the enormous catalytic potential is

essential, it does pose a problem: if it was not regulated, all reactions in a cell would rapidly reach equilibrium and, once again, life as we know it would not be possible

• Regulation of enzyme activity is achieved in a variety of ways, ranging from controls over the amount of enzyme protein produced by the cell to more rapid, reversible interactions of the enzyme with metabolic inhibitors and activators

There should be a system for the classification and naming of the several enzymes present in the cell

7

Enzyme Nomenclature• The commonly used names for most enzymes

describe the type of reaction catalyzed, followed by the suffix –ase

• For example, dehydrogenases remove hydrogen atoms, proteases hydrolyze proteins and isomerases catalyze rearrangements

• Modifiers may precede the name to indicate the substrate (lysyl oxidase), the source of the enzyme (pancreatic ribonuclease), its regulation (hormone-sensitive lipase) or a feature of its mechanism of action (cysteine protease)

• Where needed, alphanumeric designators are added to identify multiple forms of an enzyme (eg, RNA polymerase III; protein kinase Cβ).

• To address ambiguities, the International Union of Biochemists (IUB) developed an unambiguous system of enzyme nomenclature

8

• In this system, each enzyme has a unique name and code number that identify the type of reaction catalyzed and the substrates involved

• Although common names for many enzymes remain in use, all enzymes now are classified and formally named according to the reaction they catalyze

• Six classes of reactions are recognized. Within each class are subclasses, and under each subclass are subsubclasses within which individual enzymes are listed

• Classes, subclasses, subsubclasses and individual entries are each numbered, so that a series of four numbers serves to specify a particular enzyme –Enzyme Commission (EC) Number

• A systematic name, descriptive of the reaction, is also assigned to each entry

9

Classes of Enzymes1.Oxidoreductases• Oxidative reactions remove electrons, usually one

or two electrons per molecule of substrate, while reductive reactions accomplish the converse

• Oxidoreductases transfer electrons from one compound to another, thus changing the oxidation state of both substrates

• In many oxidation reduction reactions, hydrogen is transferred along with electrons

• In other reactions, a molecule or atom of oxygen could be transferred to a substance; electrons could also be transferred to oxygen

2. Transferases • Transferases catalyze reactions in which a

functional group is transferred from one compound to another

• Commonly transferred functional groups include phosphate, amino, methyl

10

3. Hydrolases • Cleave carbon-oxygen, carbon-nitrogen, carbon-

sulfur,… bonds by adding water across the bond• Digestive enzymes are hydrolases4. Lyases• Cleave carbon-oxygen, carbon-nitrogen, carbon-

sulfur,… bonds but do so without addition of water and without oxidizing or reducing the substrates

• Double bonds either arise or disappear through the action of lyases

5. Isomerases • Catalyze intramolecular rearrangements of

functional groups that reversibly interconvert optical or geometric isomers

• When an isomerase catalyzes an intramolecular rearrangement involving movement of a functional group, it is called a mutase; what is a racemase?

11

6. Ligases • Catalyze biosynthetic reactions that form a covalent

bond between two substrates.• Ligases differ from lyases in that they utilize the

energy obtained from cleavage of a high-energy bond to drive the reaction. The molecule with the high-energy bond is usually ATP• Ligases are sometimes known as synthetases and

lyases , synthases • Because the systematic names were frequently

cumbersome and the numbers difficult to memorize, the EC also proposed that a single recommended (trivial) name should be retained (or invented) for each enzyme

• For example, the enzyme catalyzing the reaction:ATP+AMP<----->2ADP

bears the systematic name ATP : AMP phosphotransferase,the number EC 2.7.4.3 and the recommended name,adenylate kinase; the earlier name, myokinase, was forsaken

12

13

14

15

16

Cofactors • Many enzymes carry out their catalytic function

relying solely on their protein structure• Many others require non-protein components,

called cofactors• Cofactors may be metal ions or organic molecules

referred to as coenzymes• Cofactors, because they are structurally less

complex than proteins, tend to be stable to heat. Typically, proteins are denatured under such conditions

• Usually coenzymes are actively involved in the catalytic reaction of the enzyme, often serving as intermediate carriers of functional groups in the conversion of substrates to products

• In many cases, a cofactor is firmly associated with its enzyme, through covalent or non-covalent bonds. Such tightly bound cofactors are referred to as prosthetic groups of the enzyme

17

• Enzymes in which metal ions serve as prosthetic groups are called metalloenzymes. Enzymes that require a non-bound metal ion cofactor are termed metal-activated enzymes

• The catalytically active complex of protein and prosthetic group is called the holoenzyme. The protein without the prosthetic group is called the apoenzyme; it is catalytically inactive

• Many coenzymes are vitamins or contain vitamins as part of their structure

• Vitamins are small organic molecules that are not synthesized in the body and are therefore essential dietary nutrients

• The vitamins that are coenzyme precursors or coenzymes include all the water-soluble B vitamins, vitamin C and the fat-soluble vitamin K

• Many coenzymes contain, in addition, the adenine, ribose and phosphoryl moieties of AMP or ADP

18

• Coenzymes are typically modified by certain reactions and are then converted back to their original forms by other enzymes; small amounts of these substances can be used repeatedly

Vitamin B1: Thiamine• It is the precursor of thiamine pyrophosphate

(TPP), a coenzyme involved in reactions where bonds to carbonyl carbons (aldehydes or ketones) are synthesized or cleaved

19

Niacin (nicotinic acid): Vitamin B3• Nicotinamide is an essential part of two

important coenzymes: nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+)

• The reduced forms of these coenzymes are NADH and NADPH

• The nicotinamide coenzymes (also known as pyridine nucleotides) are electron carriers. They play vital roles in a variety of enzyme–catalyzed oxidation–reduction reactions

• NAD+ is an electron acceptor in oxidative (catabolic) pathways and NADPH is an electron donor in reductive (biosynthetic) pathways

• These reactions involve direct transfer of hydride anion (H:) either to NAD(P) + or from NAD(P)H

• The hydride anion contains two electrons, and thus NAD + and NADP + act exclusively as two-electron carriers

20

• The C-4 position of the pyridine ring, which can either accept or donate hydride ion, is the reactive center of NAD + and NADP +

• Humans can synthesize some amount of niacin from tryptophan. However, if dietary intake of tryptophan is low, nicotinic acid is required for optimal health

• Nicotinic acid, which is beneficial to humans and animals, is structurally related to nicotine, a highly toxic tobacco alkaloid

• In order to avoid confusion of nicotinic acid and nicotinamide with nicotine itself, niacin was adopted as a common name for nicotinic acid

21

The Structures and RedoxStates of the Nicotinamide

Coenzymes

22

Riboflavin: Vitamin B2• Is a precursor of both riboflavin 5-phosphate,

also known as flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD)

• The name riboflavin is a synthesis of the names for the molecule’s component parts, ribitol and flavin

• The flavins have a characteristic bright yellow color and take their name from the Latin flavus for “yellow”

• The oxidized form of the isoalloxazine structure absorbs light around 450 nm (in the visible region) and also at 350 to 380 nm

• The color is lost, however, when the ring is reduced or “bleached”

• Similarly, the enzymes that bind flavins, known as flavoenzymes, can be yellow, red, or green in their oxidized states. These enzymes also lose their color on reduction of the bound flavin group

23

The Structures of FAD and FMN

24

• Flavin coenzymes can exist in any of three different redox states: fully oxidized flavin is converted to a semiquinone by a one-electron transfer; a second one-electron transfer converts the semiquinone to the completely reduced dihydroflavin

• The three different redox states allow flavins to participate in one-electron transfer and two-electron transfer reactions

The Oxidation States of

FAD and FMN

25

Panthotenic Acid: Vitamin B5• Makes up one part of a complex coenzyme

called coenzyme A (CoA)• Pantothenic acid is also a constituent of acyl

carrier protein (ACP)• Coenzyme A consists of 3,5-adenosine

bisphosphate joined to 4-phosphopantetheine in a phosphoric anhydride linkage

• As was the case for the nicotinamide and flavin coenzymes, CoA also contains an adenine nucleotide moiety

• CoA and ACP are involved in the activation and transfer of acyl groups

• The functions of CoA are mediated by the reactive sulfhydryl group on CoA, which forms thioester linkages with acyl groups

26

The

Stru

ctur

e of

Coe

nzym

e A

27

Pyridoxine: Vitamin B6• The biologically active form of vitamin B6 is

pyridoxal-5-phosphate (PLP) • PLP participates in the catalysis of a wide variety of

reactions involving amino acids, including transaminations, decarboxylations, racemizations and eliminations

• PLP is found as a prosthetic group attached to enzymes through a Schiff base formed between its aldehyde group and the ε-amino group of a lysine residue

28

PLP Attached to an Enzyme

The Tautomeric Forms of PLP

29

Biotin : Vitamin B7• Acts as a mobile carboxyl group carrier in a variety

of enzymatic carboxylation reactions• In each of these, biotin is bound covalently to the

enzyme as a prosthetic group via the ε-amino group of a lysine residue on the protein

• The biotin-lysine function is referred to as a biocytin residue The result is that the biotin ring system is tethered to the protein by a long, flexible chain

The Biocytin Complex

30

Folic Acid: Vitamin B9• Folic acid derivatives (folates) are acceptors and

donors of one-carbon units for all oxidation levels of carbon except that of CO2 (where biotin is the relevant carrier)

• The active coenzyme form of folic acid is tetrahydrofolate (THF)

• THF is formed via two successive reductions of folate by dihydrofolate reductase

Folic Acid

31

THF

Cyanocobalamin: Vitamin B12•Cyanocobalamin is converted in the body into two coenzymes: the predominant coenzyme form is 5-deoxyadenosylcobalamin, but smaller amounts of methylcobalamin also exist•The corrin ring, with four pyrrole groups, is similar to the heme prophyrin ring, except that two of the pyrrole rings are linked directly; iron is substituted by cobalt•There are two reactions in the body in which vitamin B12 is known to participate: a molecular rearrangement and a methyl transfer

32

Vit

amin

B12

and

its

Coen

zym

e Fo

rms

33

Ascorbic acid: Vitamin C•Has the simplest chemical structure of all the vitamins; and the coenzyme form is the vitamin itself•Ascorbic acid functions as an electron carrier. It is a strong reducing agent• It is used in the regeneration of the active form of enzymes and antioxidants

(+) H.

(-) H.

(-) 2H.

(+) 2H.

34

The Lipid-Soluble Vitamins•Vitamin K is the only fat-soluble vitamin with coenzyme role

The Vitamin A group•Vitamin A or retinol often occurs in the form of esters, called retinyl esters. The aldehyde form is called retinal or retinaldehyde•Retinol can be absorbed in the diet from animal sources or synthesized from β-carotene from plant sources•The aldehyde group of retinal forms a Schiff base with a lysine on opsin, to form light-sensitive rhodopsin

The Vitamin D group•The two most prominent members of the vitamin D family are ergocalciferol (vitamin D2) in plants and cholecalciferol (vitamin D3) in animals• Cholecalciferol is produced in the skin of animals by the action of ultraviolet light (sunlight, for example) on its precursor molecule, 7-dehydrocholesterol

35

Rhodopsin

36

•Because humans can produce vitamin D3, “vitamin D” is not strictly speaking a vitamin at all•Retinol and cholecalciferol are actually prohormones (precursors of hormones) that regulate transcription of DNA, and thus gene expression

Tocopherol: Vitamin E•α-tocopherol is a potent antioxidant; and once it has been oxidized, it can be regenerated by vitamin C

37

Naphthoquinone: Vitamin K•Clotting factors such as thrombin undergo a post-translational modification that involves the carboxylation of glutmate residues•γ-carboxyglutamyl residues are effective in the coordination of calcium, which is required for the coagulation process•The enzyme responsible for this modification, glutamyl carboxylase, requires vitamin K for its activity

The Structure of the K Vitamins

38

• Not all coenzymes are derived from vitaminsTetrahydrobiopterin (BH4), the coenzyme for

hydroxylation reactions of aromatic amino acids is synthesized in the body from GTP (guanosine triphosphate) Lipoic acid is a coenzyme used to couple acyl

transfer with electron transfer• Lipoic acid exists as a mixture of two structures: a

closed-ring disulfide form and an open–chain reduced form. Oxidation–reduction cycles interconvert these two species

• Lipoic acid is found attached with a lysine residues on enzymes

• Metal ions, which have a positive charge, contribute to the catalytic process by acting as electrophiles . They assist in binding of the substrate or they stabilize developing anions in the reaction. They can also accept and donate electrons in oxidation-reduction reactions

39The Different forms of Lipoic Acid

• Examples of metal ions and the enzymes they function with include:Zn2+ : alcohol dehydrogenase, carbonic

anhydraseMg2+ : ATP-dependent reactions such as

hexokinase Fe3+ and Cu2+: components of the enzymes of the

mitochondrial electron transport chain to the ultimate electron acceptor, oxygen

40

How Do Enzymes Work?• There are three characteristics of enzymes that

form the basis of most of their properties:1.The Active Site • In an enzyme, folding brings together amino acids,

most of which are not adjacent in the primary sequence, so that some amino acids form a three-dimensional structure that binds with the substrate to form the enzyme-substrate complex

• This complex results in catalysis• The remainder of the amino acids in the enzyme

are involved in maintenance of the three-dimensional structure of the enzyme, attaching the enzyme molecule to intracellular structures (e.g. membranes) or in binding molecules (e.g. allosteric effectors) that regulate the activity of the enzyme

2. The Enzyme-Substrate Complex • Enzymes bind substrates to produce an enzyme-

substrate complex as follows:

41

Am

ino

Acid

Sid

e-Ch

ain

Gro

ups

Invo

lved

in B

indi

ng N

AD

+ a

t th

e Ac

tive

Sit

e of

an

Enzy

me

42

E+S⇌ES• Weak bonds, generally non-covalent ones, are

involved in formation of the complex, so that the reaction is readily reversed

• The rate of the forward reaction is given by the concentration of substrate multiplied by the rate constant k1, and rate of the reverse reaction is given by the concentration of the product multiplied by the rate constant k2

• The dissociation constant for the ES complex is k2/k1

• This is analogous to the formation of other complexes: for example receptor-hormone complex; receptor-neurotransmitter complex; antibody-antigen complex

• Formation of the enzyme-substrate complex can occur only if the substrate possesses groups that are in the correct three-dimensional orientation to interact with the binding groups in the active site

k1

k2

43

• A ‘lock and key’ analogy (Emil Fischer) has been widely used to explain specificity but it is inadequate because the formation of the enzyme-substrate complex involves more than a steric complementarity between enzyme and substrate

• Enzymes are highly flexible, conformationally dynamic molecules, and many of their remarkable properties, including substrate binding and catalysis, are due to this flexibility

• Realization of the conformational flexibility of proteins led Daniel Koshland to hypothesize that the binding of a substrate by an enzyme is an interactive process

• The shape of the enzyme’s active site is actually modified upon binding S, in a process of dynamic recognition between enzyme and substrate called ‘induced fit’

• Substrate binding alters the conformation of the protein, so that the protein and the substrate “fit” each other more precisely. The conformation of the substrate also changes as it adapts to the conformation of the enzyme

44

3.The Transition State• In a chemical reaction, one stable arrangement of

atoms (the substrate) is converted to another (the product)

• As this change proceeds, the atoms pass through an unstable arrangement, known as the transition state, which can be thought of as the ‘halfway house’ between the substrates and the products

• The relevance of the transition state to kinetics is that the rate of the overall reaction depends on the number of molecules in this state: the more molecules in the transition state, the greater is the rate

• The role of an enzyme is to increase the number of molecules in this state

• Enzymes increase the number of molecules in the transition state through one or more of five mechanisms

45

The Energy Level and the StructuralFeature of the Transition State

46

Mechanisms of Enhancing the Rate of a Reaction

• Since the transition state possesses the least stable electron distribution, an agent capable of supplying or withdrawing electrons to or from stable parts of a substrate in order to destabilize it, accelerates the rate of a reaction

1. General acid base catalysis• Addition of a proton from an acid to a molecule

can cause an electron to be withdrawn from one part of the molecule to the part which binds the proton

• A base removes a proton from a molecule which will also cause electron shifts

• If these shifts favor the formation of the transition state, the rate of the reaction increases

• The active sites of enzymes possess side-chain groups of amino acids that act as acids or bases

47

• The contribution of these groups is greatly enhanced if they act in a concerted manner so that, as an electron is withdrawn from one part of the substrate, another is donated to a different part

• This is possible only when the relevant groups in the active site are held in precisely the correct orientation so as to interact in this way with the substrate

• One of the more versatile side-chains in this respect is the imidazole group of histidine. In one environment it can act as an acid whereas, in another environment, the same group can act as a base

• This can occur with two histidines in the same active site

• Acid base catalysis occurs on the vast majority of enzymes. In fact, proton transfers are the most common biochemical reactions

Specific acid base catalysis occurs when only the protons and hydroxyls present in solution are used

48

Am

ino

Acid

s In

volv

ed in

Aci

d B

ase

Cata

lysi

s

49

2. Covalent catalysis (formation of an intermediate)

• Most enzymes bind their substrates in a non-covalent manner but, for those that do bind covalently, the intermediate must be less stable than either substrate or product

• Many of the enzymes that involve covalent catalysis are hydrolytic enzymes; these include proteases, lipases, phosphatases and also acetylcholinesterase

• A number of amino acid side chains, including all those that participate in acid base catalysis and the functional groups of some coenzymes can serve as nucleophiles in the formation of covalent bonds with substrates. These covalent complexes always undergo further reaction to regenerate the free enzymeAn Example of Covalent Catalysis

50

An Example of Covalent Catalysis

3. Metal Ion Catalysis• Metals, whether tightly bound to the enzyme or

taken up from solution along with the substrate, can participate in catalysis in several ways

• Ionic interactions between an enzyme-bound metal and a substrate can help orient the substrate for reaction or stabilize charged reaction transition states

• Metals can also mediate oxidation-reduction reactions by reversible changes in the metal ion’s oxidation state

• Nearly a third of all known enzymes require one or more metal ions for catalytic activity

51

4. Proximity and Orientation • In a reaction involving two substrates, the two

must come together in order to react• The chance of them doing so depends upon

their concentration in the solution: this is increased locally by providing adjacent binding sites for each substrate within the active site

• This can increase the effective concentrations of the substrates about 1000-fold

• Even when a collision between two substrates occurs it is unlikely that they will both be in the correct orientation for a reaction to take place

• Another property of the active site is that it binds the substrates in such a way that their orientation favors the reaction, i.e., it facilitates electron shifts that favor formation of the transition state

52

5. Strain (Distortion)• Enzymes that catalyze -lytic reactions that involve

breaking a covalent bond typically bind their substrates in a conformation slightly unfavorable for the bond that will undergo cleavage

• The resulting strain stretches or distorts the targeted bond, weakening it and making it more vulnerable to cleavage

The Catalytic Mechanism of the Aspartic Protease Family

(Example of Acid Base Catalysis)• Enzymes of the aspartic protease family, which

includes the digestive enzyme pepsin, the lysosomal cathepsins and the protease produced by HIV share a common catalytic mechanism

• Catalysis involves two conserved aspartyl residues, which act as acid-base catalysts

• In the first stage of the reaction, an aspartate functioning as a general base (Asp X) extracts a proton from a water molecule, making it more nucleophilic

53

• The resulting nucleophile then attacks the electrophilic carbonyl carbon of the peptide bond targeted for hydrolysis, forming a tetrahedral transition state intermediate

• A second aspartate (Asp Y) then facilitates the decomposition of this tetrahedral intermediate by donating a proton to the amino group produced by rupture of the peptide bond

• The two different active site aspartates can act simultaneously as a general base or as a general acid because their immediate environment favors ionization of one, but not the other

54

55

Carbonic Anhydrase and Metal Ion Catalysis• A zinc prosthetic group in carbonic anhydrase is

coordinated in three positions by histidine side-chains. The fourth coordination position is occupied by water

• Binding of water to zinc reduces pKa of water from 15.7 to 7

• Zinc facilitates the release of a proton from a water molecule, which generates a hydroxide ion that can act as a nucleophile

• The carbon dioxide substrate binds to the enzyme's active site and is positioned to react with the hydroxide ion

• The hydroxide ion attacks the carbon dioxide, converting it into bicarbonate ion

56

• The catalytic site is regenerated with the release of the bicarbonate ion and the binding of another molecule of water

The Catalytic Mechanism of Carbonic Anhydrase

57

Factors that Change the Activity of an Enzyme• The main factors that can change the catalytic

activity of an enzyme are concentrations of substrates, pH, temperature and inhibitors

• The effects of these factors and the means by which they are studied are usually described as enzyme kinetics

The Effect of Substrate Concentration • The activity or rate of an enzyme (v) varies

according to the substrate concentration [S]. The relationship is hyperbolic:

At very low substrate concentrations, the reaction

rate is approximately first order (i.e. activity increases approximately linearly with increase in substrate concentration)

At very high substrate concentrations, the rate of reaction approaches zero order (i.e. the increase in substrate concentration has very little effect on the rate of reaction)

58

This behavior is a saturation effect: when v shows no increase even though [S] is increased, the system is saturated with substrate. The physical interpretation is that every enzymemolecule in the reaction mixture has its substrate-binding site occupied by S At intermediate substrate concentrations, the

order is intermediate between zero and first order

Even though enzymes with a single substrate are considered here, the same principles apply to enzymes with more than one substrate

59

• A hyperbolic curve is described by an equation of the form:

• In the case of an enzyme,

where a and b are constants • Two questions:

1. What is the mechanism of catalysis that accounts for the hyperbolic relationship?

2.What are the constants a and b?• These questions were answered by the works of

Michaelis and Menten & Briggs and Haldane• Michaelis and Menten’s theory was based on the

assumption that the enzyme, E, and its substrate, S, associate reversibly to form an enzyme-substrate complex, ES:

60

• This association/dissociation is assumed to be a rapid equilibrium (hence the equilibrium model), and Ks is the enzyme : substrate dissociation constant. At equilibrium,

• Product, P, is formed in a second step when ES breaks down to yield EP; this step is slower

• E is then free to interact with another molecule of S

61

• The interpretations of Michaelis and Menten were refined by Briggs and Haldane, who postulated that the concentration of ES quickly reaches a constant value

• ES is formed as rapidly from E+S as it disappears by its two possible fates: dissociation to regenerate E+S, and reaction to form E+P

• This assumption is termed the steady-state assumption and is expressed as

• Two additional assumptions are made: In the reaction mixture, [S] is greater than

[E].However, [S] is not so large that all enzyme molecules are in the ES form, but [S] must be sufficiently large that [S] does not rapidly become so small that [S] < [E]

Because enzymes accelerate the rate of the reverse reaction as well as the forward reaction, it would be helpful to ignore any back reaction by which EP might form ES

62

The velocity of this back reaction would be given by v=k-2[E][P]. However, if only the initial velocity for the reaction (immediately after E and S are mixed in the absence of P) is measured, the rate of any back reaction is negligible because its rate will be proportional to [P], and [P] is essentially 0• The total amount of enzyme is fixed and is given by

the formula total enzyme, [ET]=[E]+[ES] where [E] = free enzyme and [ES]=the amount of enzyme in the enzyme– substrate complex• The rate of [ES] formation is

• The rate of disappearance of ES

• At the steady state, rate of formation=rate of disappearance

• Rearranging gives

63

• The ratio of constants (k-1+k2)/k1 is itself a constant and is defined as the Michaelis constant, Km• Because it is given as a ratio of concentrations, the

unit of Km is molarity • Rearranging the equation for the derivation of Km

• The rate of product formation is given asv=k2[ES]

• Substituting for the value of [ES]

64

• The product k2[ET] has a special meaning. When [S] is high enough to saturate all of the enzyme, the velocity of the reaction, v, is maximal• At saturation, the amount of [ES] complex is

equal to the total enzyme concentration, ET, its maximum possible value. • The initial velocity, v, then equals k2[ET]=Vmax

• This equation states that the rate of an enzyme-catalyzed reaction, v, at any moment is determined by two constants, Km and Vmax, and the concentration of substrate at that moment• When S= Km , V= Vmax/2 : Km is the substrate

concentration that gives a velocity equal to one-half the maximal velocity

The Michaelis-Menten Equation

65

• In the rectangular hyperbola, as [S] is increased, v approaches the limiting value, Vmax, in an asymptotic fashion• When [S]>>Km, then v=Vmax. That is, v is no longer

dependent on [S], so the reaction is obeying zero-order kinetics• When [S]<<Km, then v≈(Vmax/Km)[S]. That is v

approximately follows a first-order rate equation, v=k’[S], where k’ =Vmax/Km

66

The Features of Vmax and Km

• Km is a constant for a particular enzyme and substrate and is independent of enzyme and substrate concentrations.• Vmax depends on enzyme concentration, and at

saturating substrate concentration, it is independent of substrate concentration.• Km and Vmax may be influenced by pH, temperature

and other factors • When k-1 >> k2 (when the formation of product is

the rate limiting step), [ES] is assumed to be at equilibrium with [E] and [S] ,i.e, ES is dissociating more often to yield E and S than to yield product• Under this condition, Km is simplified to k-1 /k1 which

in turn is equal to the dissociation constant , Ks. Where these conditions hold, Km does represent a measure of the affinity of the enzyme. for its substrate in the ES complex. However, this scenario does not apply for most enzymes

67

• When [S] >> Km, the characteristic property of the turnover number for an enzyme can be used• This number provides information on how many

times the enzyme performs its catalytic function per unit time, or how many times it forms the ES complex and is regenerated (turned over) by yielding product• The rate-limiting step of the enzymatic reaction can

give a good indicator of the turnover number, and hence, the kinetic efficiency • Vmax=k2[ES]=k2[ET]• k2 is denoted as kcat and gives the value of the

turnover number• Catalase has the highest turnover number known;

each molecule of this enzyme can degrade 40 million molecules of H2O2 in one second. At the other end of the scale, lysozyme requires 2 seconds to cleave a bond in its substrate• Under physiological conditions, [S] is seldom

saturating, and kcat itself is not particularly informative

68

• The in vivo ratio of [S]/Km usually falls in the range of 0.01 to 1.0, so active sites often are not filled with substrate• A meaningful index of the efficiency of enzymes

under these conditions could be derived as follows:

• When S<<Km the concentration of free enzyme, [E], is approximately equal to [ET], so that

• kcat/Km is an apparent second-order rate constant for the reaction of E and S to form product • Because Km is inversely proportional to the affinity

of the enzyme for its substrate and kcat is directly proportional to the kinetic efficiency of the enzyme, kcat/Km provides an index of the catalytic efficiency of an enzyme operating at substrate concentrations substantially below saturation amounts

69

• The ratio kcat/Km can be expressed as

• But k1 must always be greater than or equal to k1k2/(k-1+k2). That is, the reaction can go no faster than the rate at which E and S come together• Thus, k1 sets the upper limit for kcat/Km. In other

words, the catalytic efficiency of an enzyme cannot exceed the diffusion-controlled rate of combination of E and S to form ES• In water , the rate constant for such diffusion is

approximately 109/M.sec• Those enzymes that are most efficient in their

catalysis have kcat/Km ratios approaching this value. Their catalytic velocity is limited only by the rate at which they encounter S; enzymes this efficient have achieved so-called catalytic perfection• All E and S encounters lead to reaction because

such enzymes can channel S to the active site, regardless of where S hits E

70

Linear Plots for the Michaelis Menten Equation• Vmax can be approximated experimentally from a

substrate saturation curve and Km can be derived from Vmax/2, so the two constants of the Michaelis–Menten equation can be obtained from plots of v versus [S]• However, straight-line plots are easier to evaluate

than curves, and the Michaelis Menten equation is reformulated to yield straight-line plots• The best known of these reformulations is the

Lineweaver–Burk double-reciprocal plot• Taking the reciprocal of both sides of the MM

equation gives

• This conforms to y=mx+b (the equation for a straight line), where y=1/v; m, the slope, is Km/Vmax; x=1/[S]; and b=1/Vmax. The x-intercept of the line is -1/Km and y-intercept is 1/Vmax

71

The

Line

wea

ver-

Bur

k Pl

ot

The Effect of pH on Enzymatic Activity • The pH-enzyme activity profile of most enzymes

can be depicted by a bell-shaped curve, exhibiting an optimal pH at which activity is maximal• The optimal pH is usually the same as the pH of

the fluid in which the enzyme functions. Thus, most enzymes in the body have their highest activity between pH 6 and pH 8 (the pH of human blood is about 7.4)

72

• However, pepsin, which must function at the low pH of gastric juice, has maximal activity at about pH 2• An enzyme possesses many ionizable side chains and

prosthetic groups that not only determine its secondary and tertiary structure but that are actively involved in its active site• Further, the substrate itself often has ionizing groups,

and one or another of the ionic forms may preferentially interact with the enzyme. Changes in pH affect the binding of the substrate at the active site of the enzyme and also the rate of breakdown of the enzyme-substrate complex

The

pH

Acti

vity

Pr

ofile

s of

Fou

r En

zym

es

73

The Effect of Temperature on Enzymatic Activity

• Raising the temperature increases the rate of both uncatalyzed and enzyme-catalyzed reactions by increasing the kinetic energy and the collision frequency of the reacting molecules• Most enzymatic reactions double in rate for every

10°C rise in temperature• However, heat energy can also increase the kinetic

energy of the enzyme to a point that exceeds the energy barrier for disrupting the non-covalent interactions that maintain its three-dimensional structure• The polypeptide chain then begins to unfold, or

denature, with an accompanying loss of catalytic activity• The temperature range over which an enzyme

maintains a stable, catalytically active conformation depends upon—and typically moderately exceeds—the normal temperature of the cells in which it resides

74

• Enzymes from humans generally exhibit stability at temperatures up to 45–55°C• By contrast, enzymes from the thermophilic

microorganisms that reside in volcanic hot springs or undersea hydrothermal vents may be stable up to or even above 100°C• For mammals and other homeothermic organisms,

changes in enzyme reaction rates with temperature assume physiologic importance only in circumstances such as fever or hypothermia

The

Eff

ect

of

Tem

pera

ture

on

Enzy

me

Acti

vity

75

Reactions of Two or More Substrates• Enzymes frequently catalyze the reaction of two, three or even more different molecules to give one, two, three or more products• Sometimes all of the substrate molecules must be bound to an active site at the same time and are presumably lined up on the enzyme molecule in such a way that they can react in proper sequence• In other cases, the enzyme may transform molecule A to a product, and then cause the product to react with molecule B. The number of variations is enormous•Two common types of two-substrate, two-product reactions (termed “Bi-Bi” reactions) are sequential and ping pong reactions

Sequential or Single-Displacement Reactions • In sequential reactions, both substrates must combine with the enzyme to form a ternary complex before catalysis can proceed

76

•Sequential reactions are sometimes referred to as single-displacement reactions because the group undergoing transfer is passed directly, in a single step, from one substrate to the other•Sequential reactions can be of two distinct types:a. random, where either A or B may bind to the

enzyme first, followed by the other substrate

b. ordered, where A, designated the leading substrate, must bind to E first before B can be boundOne explanation for an ordered mechanism is

that the addition of A induces a conformational change in the enzyme that aligns residues that recognize and bind B

Random

77

Ordered

Ping Pong Reactions• The term “ping-pong” applies to mechanisms in

which one or more products are released from the enzyme before all the substrates have been added

• Ping-pong reactions involve covalent catalysis and a transient, modified form of the enzyme

• Ping-pong Bi-Bi reactions are double displacement reactions. The group undergoing transfer is first displaced from substrate A by the enzyme to form product P and a modified form of the enzyme (F). The subsequent group transfer from F to the second substrate B, forming product Q and regenerating E, constitutes the second displacement

78

Ping Pong

Enzyme Inhibition • Enzyme inhibition is one of the ways by which enzyme activity is regulated experimentally or naturally•Several therapeutic drugs function by inhibition of a specific enzyme (of humans or pathogens)• Inhibitor studies have contributed much of the available information about enzyme kinetics and mechanisms•Reversible inhibitors interact with an enzyme through non-covalent association/dissociation reactions• In contrast, irreversible inhibitors usually cause stable, covalent alterations in the enzyme

79

•Reversible inhibition is further divided into three types:

1. Competitive Inhibition• In this type of reversible inhibition, a compound

competes with an enzyme’s substrate for binding to the active site,

• This results in an apparent increase in the enzyme–substrate dissociation constant (Ks ) (i.e., an apparent decrease in the affinity of enzyme for substrate) without affecting the enzyme’s maximum velocity (Vmax)

80

•The rate equation for the formation of product, the dissociation constants for enzyme–substrate (ES) and enzyme–inhibitor (EI) complexes and the enzyme mass balance are, respectively:

•Normalization of the rate equation by total enzyme concentration (v/[ET ]) and rearrangement results in the following expression for the velocity of an enzymatic reaction in the presence of a competitive inhibitor:

81

• Ks∗ corresponds to the apparent enzyme–substrate

dissociation constant in the presence of an inhibitor. In the case of competitive inhibition, Ks

∗

=αKs• Competitive inhibition can be relieved by

increasing the concentration of substrate• Examples of competitive inhibition:A classic example is the inhibition of succinate

dehydrogenase by succinate’s analogues malonate, oxalate or oxaloacetate

82

p-aminobenzoic acid is required by bacteria for the synthesis of folic acid which functions as a coenzyme in one-carbon transfer reactions that are important in amino acid metabolism, in the synthesis of RNA and DNA and thus in cell growth and division. Sulfanilamides are analogues of PABA that inhibit the synthesis of folic acid

2.Uncompetitive Inhibition • In this type of reversible inhibition, a compound

interacts with the enzyme– substrate complex at a site other than the active site

• This results in an apparent decrease in both Vmax and Ks

83

• The apparent increase in affinity of enzyme for substrate (i.e., a decrease in Ks ) is due to unproductive substrate binding, resulting in a decrease in free enzyme concentration• Half-maximum velocity, or half-maximal saturation,

will therefore be attained at a relatively lower substrate concentration• The rate equation for the formation of product, the

dissociation constants for enzyme–substrate (ES) and ES–inhibitor (ESI) complexes and the enzyme mass balance are, respectively

84

• Normalization of the rate equation by total enzyme concentration (v/[ET ]) and rearrangement results in the following expression:

• Vmax∗ and Ks

∗ correspond, respectively, to the apparent enzyme maximum velocity and apparent enzyme–substrate dissociation constant in the presence of an inhibitor• In the case of uncompetitive inhibition, Vmax

∗= Vmax/α and Ks

∗= Ks/α

85

3.Mixed Inhibition• In this type of reversible inhibition, a compound can

interact with both the free enzyme and the enzyme–substrate complex at a site other than the active site:

• This results in an apparent decrease in Vmax and an apparent increase in Ks

• The rate equation for the formation of product, the dissociation constants for enzyme–substrate (ES and ESI) and enzyme– inhibitor (EI and ESI) complexes, and the enzyme mass balance are, respectively,

86

• Normalization of the rate equation by total enzyme concentration (v/[ET ]) and rearrangement results in the following expression:

• In the case of mixed inhibition, Vmax∗= Vmax/β and

Ks∗=(α/ β)Ks

87

Non-Competitive Inhibition is a special case of mixed inhibition

where δ = 1 and α = β. Thus, the expression for the velocity of an enzymatic reaction in the presence of a non-competitive inhibitor becomes:

• Thus, for non-competitive inhibition, an apparent decrease in Vmax is observed while Ks remains unaffected

A Comparison of the Effects of Different Types of Inhibitors

Com

peti

tive

Inhi

biti

on

Uncompetitive Inhibitio

n

88

Mix

ed In

hibi

tion • In the cases of

uncompetitive and mixed inhibition, the activity of the enzyme is fully restored when the inhibitor is removed from the system (by dialysis, gel filtration or other separation techniques) in which the enzyme functions

Irreversible Inhibition• Irreversible inhibition occurs when the inhibitor

reacts at or near the active site of the enzyme with covalent modification of the active site or when the inhibitor binds so tightly that, there is no dissociation of enzyme and inhibitor

• Thus, physical separation processes are ineffective in removing the irreversible inhibitor from the enzyme

89

•The serine protease family comprises of enzymes such as chymotrypsin, trypsin, acetylcholinesterase and thrombin; acetylcholinesterase shows a similar mechanism and is a serine esterase •The essential serine residues in these enzymes can be inhibited by different agents resulting in inactivation•The lethal compound diisopropyl phosphofluoridate (DIFP) is an organophosphorus compound that served as a prototype for the development of the nerve gas sarin and other organo-phosphorus toxins, such as the insecticides malathion and parathion •DIFP exerts its toxic effect by forming a covalent intermediate in the active site of acetylcholinesterase, thereby preventing the enzyme from degrading the neurotransmitter acetylcholine• Once the covalent bond is formed, the inhibition by DIFP is essentially irreversible, and activity can only be recovered as new enzyme is synthesized

90

91

•Aspirin (acetylsalicylic acid) provides an example of a pharmacologic drug that exerts its effect through the covalent acetylation of an active site serine in the enzyme prostaglandin endoperoxide synthase (cycloxygenase)• Aspirin resembles a portion of the prostaglandin precursor that is a physiologic substrate for the enzyme

92

•Mechanism-based inhibitors are a group made up of irreversible inhibitors and transition state analogs •Transition state analogs are extremely potent and specific inhibitors of enzymes because they bind so much more tightly to the enzyme than do substrates or products• Drugs cannot be designed that precisely mimic the transition state because of its highly unstable structure•However, substrates undergo progressive changes in their overall electrostatic structure during the formation of a transition state complex, and effective drugs often resemble an intermediate stage of the reaction more closely than they resemble the substrate•Such compounds are often referred to as substrate analogs, even though they bind more tightly than substrates•The antibiotic penicillin is a transition state analog that binds very tightly to glycopeptidyl transferase, an enzyme required by bacteria for synthesis of the cell wall

93

•Glycopeptidyl transferase catalyzes a partial reaction with penicillin that covalently attaches penicillin to its own active site serine• The reaction is favored by the strong resemblance between the peptide bond in the β-lactam ring of penicillin and the transition state complex of the natural transpeptidation reaction•Active site inhibitors such as penicillin that undergo partial reaction to form irreversible inhibitors in the active site are sometimes termed “suicide inhibitors” or “Trojan horse substrates”

94

Enzyme Regulation• The metabolic rate of key substances, which can

proceed in multiple pathways, is regulated and integrated

• This regulation and integration is the result of the control exerted over the activity of enzymes

• A metabolic pathway involves many enzymes functioning in an ordered manner to carry out a particular metabolic process

• Control of a pathway is accomplished through modulation of the activity of only one or a few key enzymes

• These regulatory enzymes usually catalyze the first or an early reaction in a metabolic sequence

• A regulatory enzyme catalyzes a rate-limiting (or rate-determining) chemical reaction that controls the overall pathway. It may also catalyze a chemical reaction unique to that pathway, which is known as a committed step

• The rate-limiting step need not be the same as the committed step

95

• Those enzymes which catalyze the rate-limiting step or the committed step of a pathway are under regulation

• When the a product exceeds the steady-state level concentration, it inhibits the regulatory enzyme in an attempt to normalize the overall process. This type of control is known as feedback inhibition

• Regulation can be based upon two main aspects of enzymes –their amounts and/or activities

Control Over Amount • There are genetic controls over the amounts of

enzyme synthesized by cells• If the gene encoding a particular enzyme protein

is turned on or off, changes in the amount of enzyme activity soon follow

• Induction, which is the activation of enzyme synthesis, and repression, which is the shutdown of enzyme synthesis, are important mechanisms for the regulation of metabolism

96

• By controlling the amount of an enzyme that is present at any moment, cells can either activate or terminate various metabolic routes

• Genetic controls over enzyme levels have a response time ranging from minutes in rapidly dividing bacteria to hours (or longer) in higher eukaryotes

• In addition to the regulation of the rate of synthesis of enzymes, the degradation of enzymes induced by physical and/or chemical changes in the enzyme, may determine the amount of enzyme present

Control Over Catalytic Activity • Changes in intrinsic catalytic efficiency effected

by binding of dissociable ligands (allosteric regulation), covalent modification, proteolytic cleavage or protein-protein interaction achieve regulation of enzymatic activity within seconds

97

• Changes in protein level serve long-term adaptive requirements, whereas changes in catalytic efficiency are best suited for rapid and transient alterations in metabolite flux

Allosteric Regulation • Those enzymes in metabolic pathways whose

activities can be regulated by non-covalent interactions of certain compounds at sites other than the catalytic are known as allosteric enzymes

• The term "allosteric" is of Greek origin, the root word "allos" meaning "other’’

• Thus, an allosteric site is a unique region of an enzyme other than the substrate binding site that leads to catalysis

• At the allosteric site, the enzyme is regulated by non-covalent interaction with specific ligands known as effectors or modulators

98

• Allosteric enzymes are characterized by cooperativity: the binding of a ligand to an allosteric site affects binding of the substrate to the enzyme

• Binding of an allosteric modulator causes a change in the conformation of the enzyme that leads to a change in the binding affinity of the enzyme for the substrateAllosteric enzymes are those having “other

shapes” or conformations induced by the binding of modulators

• The catalytically more active conformation of allosteric enzymes is known as the relaxed or R-state or R-state; the less active conformation is known as the taut or T-state

• The effect of a modulator may be positive (activatory) or negative (inhibitory). The former leads to increased affinity of the enzyme for its substrate, whereas the reverse is true for the latter. Activatory sites and inhibitory sites are separate and specific for their respective modulators

• Feedback inhibitors are negative allosteric regulators

99Subunits Interactions in Allosteric Enzymes

100

• Two types of interaction occur in allosteric enzymes: homotropic and heterotropic

• In a homotropic interaction, the same ligand influences positively the cooperativity between different modulator sites. Heterotropic interaction refers to the effect of one ligand on the binding of a different ligand

• Allosteric modulators should not be confused with uncompetitive and mixed inhibitors

• Although the latter bind at a second site on the enzyme, they do not necessarily mediate conformational changes between active and inactive forms, and the kinetic effects are distinct

• Allosteric enzymes are generally larger and more complex than non-allosteric enzymes. Most have two or more subunits

• For example, aspartate transcarbamoylase (ATCase), which catalyzes an early reaction in the biosynthesis of pyrimidine nucleotides, has 12 polypeptide chains organized into catalytic and regulatory subunits

101

• Allosteric enzymes show relationships between V and [S] that differ from Michaelis-Menten kinetics

• For homotropic allosteric enzymes, plots of V versus [S] produce a sigmoid saturation curve, rather than the hyperbolic curve typical of non-regulatory enzymes

• Sigmoid kinetic behavior generally reflects cooperative interactions between protein subunits

• Changes in the structure of one subunit are translated into structural changes in adjacent subunits, an effect mediated by non-covalent interactions at the interface between subunits

• On the sigmoid saturation curve the value of [S] at which V is half-maximal is referred to as K0.5(instead of Km)

• For heterotropic allosteric enzymes, it is difficult to generalize about the shape of the substrate-saturation curve

• An activator may cause the curve to become more nearly hyperbolic, with a decrease in K0.5 but no change in Vmax

102

• Other heterotropic allosteric enzymes respond to an activator by an increase in Vmax with little change in K0.5

• A negative modulator may produce a more sigmoid substrate-saturation curve, with increase in K0.5 or decrease in Vmax

The Sigmoid Curve of a Homotropic Enzyme

103

The Activity Curves of Heterotropic Allosteric Enzymes

104

Covalent Modification• In another important class of regulatory enzymes,

activity is modulated by covalent modification of the enzyme molecule

• Modifying groups include phosphoryl, adenylyl, uridylyl, methyl and adenosine diphosphate ribosyl groups

• Phosphorylation is the most common type of regulatory modification; one-third to one-half of all proteins in a eukaryotic cell are phosphorylated

• Some proteins have only one phosphorylated residue, others have several and a few have dozens of sites for phosphorylation

• The attachment of phosphoryl groups to specific amino acid residues of a protein is catalyzed by protein kinases; removal of phosphoryl groups is catalyzed by phosphoprotein phosphatases

• Phosphoprotein phosphatases show less substrate specificity than protein kinases

105

• The addition of a phosphoryl group to a Ser, Thr or Tyr residue introduces a bulky, charged group into a region that was only moderately polar

• The oxygen atoms of a phosphoryl group can hydrogen-bond with one or several groups in a protein, commonly the amide groups of the peptide backbone or an arginine residue

• The two negative charges on a phosphorylated side chain can also repel neighboring negatively charged (Asp or Glu) residues

• When the modified side chain is located in a region of the protein critical to its three dimensional structure, phosphorylation can have dramatic effects on protein conformation and thus on substrate binding and catalysis

106Exam

ples

of

Cova

lent

Mod

ifica

tion

107

Proteolytic Activation (and Protein-Protein Interaction)

• Most proteins become fully active as their synthesis is completed and they spontaneously fold into their native, three-dimensional conformations

• Some proteins,however, are synthesized as inactive precursors, called zymogens or proenzymes, that only acquire full activity upon specific proteolytic cleavage of one or several of their peptide bonds

• Unlike allosteric regulation or covalent modification, zymogen activation by specific proteolysis is an irreversible process

• The synthesis of zymogens as inactive precursors prevents them from cleaving proteins prematurely at their sites of synthesis or secretion

• Chymotrypsinogen, for example, is stored in vesicles within pancreatic cells until secreted into ducts leading to the intestinal lumen. In the digestive tract, chymotrypsinogen is converted to chymotrypsin by the proteolytic enzyme trypsin

108

• Trypsin cleaves off a small peptide from the N-terminal region (and two internal peptides)

• This cleavage activates chymotrypsin by causing a conformational change in the spacing of amino acid residues around the binding site for the denatured protein substrate and around the catalytic site

• Most of the proteases involved in blood clotting are zymogens, such as fibrinogen and prothrombin, which circulate in blood in the inactive form

• They are cleaved to the active form (fibrin and thrombin, respectively) by other proteases, which have been activated by their attachment to the site of injury in a blood vessel wall

• Thus, clots form at the site of injury and not randomly in circulation

• Because proteolytic activation is irreversible, a way of inactivating enzymes is needed

109

110

• Most proteolytic enzymes of the digestive and the blood coagulation system contain serine in the active centre and are inhibited by the binding of serine protease inhibitors (serpins)

• There are more than 40 serpins in blood and they play an important role in inhibition of some powerful proteolytic enzymes, e.g. those involved in blood clotting

• One of the best-known examples of a serpin is α1-antiproteinase (antitrypsin), which inhibits elastase. Elastase is a proteolytic enzyme that is secreted by macrophages, among other cells

• Elastase is one of the many weapons used by macrophages to kill invading pathogens. The macrophages are particularly important in the lung, since it is a relatively easy point of entry for pathogens

• However, once the pathogen has been killed, elastase activity must be inhibited and this is achieved by release of α1-antitrypsin

111

• The free radicals in tobacco smoke cause chronic damage to the cells of the lung; they also decrease the affinity of α1-antiproteinase for elastase. The resultant damage to the lung tissue usually leads to emphysema

• Another example of serpins is, pancreatic trypsin inhibitor, which binds to and inhibits trypsin

• In contrast with nearly all known protein assemblies, the trypsin-pancreatic trypsin inhibitor complex is not dissociated into its constituent chains by treatment with denaturing agents such as 8 M urea or 6 M guanidine hydrochloride

o Some protein hormones are synthesized in the form of inactive precursor molecules, from which the active hormone is derived by proteolysis. For instance, insulin is generated by proteolytic excision of a specific peptide from proinsulin

o Collagen also is initially synthesized as the soluble precursor procollagen

112

Clinical Applications of Enzymes• When a tissue is damaged, infected or inflamed,

cell membranes become more permeable or are destroyed so that the content of the cytoplasm, especially the dissolved substances, are able to pass to the extracellular space and then into the bloodstream

• The blood can be drawn, plasma can be prepared and the enzymatic activity can be measured

• These measurements have been useful, particularly in the diagnosis of damage to the heart, liver and muscle, and enzyme measurements are useful in following the healing process and the prognosis

• Plasma-specific enzymes are enzymes that are normally present in plasma, perform their primary function in blood and have levels of activity that are usually higher in plasma than in tissue cells. Examples are enzymes involved in blood clotting and immune response

113

• Non-plasma-specific enzymes are intracellular enzymes normally present in plasma at minimal levels or at concentrations well below those in tissue cells

• Their presence in plasma is normally due to turnover of tissue cells, but they are released into the body fluids in excessive concentrations as a result of cellular damage or impairment of membrane function

• Normal cell turnover is carried out through a process known as apoptosis (from the Greek for “shedding’’)

• Apoptosis is a programmed cell death which may be initiated when cells fail to receive a life-maintaining signal (suicide) or when they receive a death signal (murder)

• Cells dying by apoptosis undergo characteristic morphological changes: they shrink and condense; the cytoskeleton collapses; the nuclear envelope disassembles; and the nuclear chromatin condenses and breaks into fragments

114

• The cell surface often blebs and breaks up into membrane-enclosed fragments called apoptotic bodies

• The apoptotic bodies are engulfed by neighboring cells or macrophages before they can spill their contents

• In this way, the cell dies neatly and is rapidly cleared away, without causing a damaging inflammatory response

• In contrast to apoptosis, cells that die accidentally, in response to an acute insult, usually do so by a process called cell necrosis (from Greek for “corpse”)

• Necrotic cells swell and burst, spilling their contents over their neighbors and eliciting an inflammatory response

• The enzymes that are measured in clinical laboratories are those that are released from necrotic cells

• Certain enzymes have been of interest over the years

115

116

• The enzymes concentrated in the heart and liver are aspartate aminotransferase (also called serum glutamate-oxaloacetate transaminase (SGOT)) and alanine aminotransferase (also called serum glutamate-pyruvate transaminase (SGPT))

• Alkaline phosphatase (AP) is reflective of bone, intestine and other tissues. Creatine kinase (CK) is reflective of skeletal and cardiac muscle

• Lactate dehydrogenase (LDH) is reflective of heart, liver, muscle and red blood cells

• α- amylase reflects the pancreas and acid phosphatase reflects the prostate gland

• By measuring different enzyme activities, a pattern is seen that characterizes an organ

• However, the search can be made more specific and defining when the isoenzymes/isozymes, if present, are measured

117

• Isozymes are quaternary forms of an enzyme differing in their relative proportions of structurally equivalent but catalytically distinct polypeptide subunits

• A classic example of isozymes is mammalian LDH which exists as five different isozymes, depending on the tetrameric association of two different subunits, H (heart-type) and M (skeletal-muscle-type): H4, H3M, H2M2, HM3 and M4

• The kinetic properties of the various LDH isozymes differ in terms of their relative affinities for the various substrates and their sensitivity to inhibition by product

• Another example could be CK, which is a dimer made up of M (muscle-type) subunit and B (brain-type) subunit

• Consequently, there are three CK isozymes: MM, MB and BB

• Different tissues express different isozyme forms, as appropriate to their particular metabolic needs

118