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Enzymes

Subtopics

Definition of enzymes

Properties of enzymes

Major classes of enzymes

Enzyme kinetics

Enzyme mechanism (mechanism of catalysis)

Regulation of enzyme activity (activation/inhibition)

Enzymology

Nearly all biochemical reactions that comprise life do not take place at perceptible rates in the absence

of enzymes. Enzymes are remarkable biological catalysts. They are also involved in energy transmission

and energy transformation. They can transform energy from one form into another. During

photosynthesis, light energy is converted into chemical-bond energy whereas during cellular

respiration, which takes place in mitochondria, the free energy contained in small molecules derived

from food is converted first into the free energy of an ion gradient and then into a different currency—

the free energy of adenosine triphosphate. Nearly all known enzymes are proteins with the exception of

catalytically active RNA molecules (Ribozymes). RNA might be the biocatalyst early in evolution. The

most striking characteristics of enzymes are their catalytic power and specificity.

Enzymes differ from ordinary chemical catalysts:

Higher reaction rates: the rates of enzyme-catalyzed reactions are several orders of magnitude

greater than those of chemical-catalyzed reactions.

Milder reaction conditions: enzyme-catalyzed reactions occur under relatively mild conditions:

temperatures below 100°C, atmospheric pressure, and nearly neutral pH’s

Greater reaction specificity: Enzymes are more specific with respect to the identities of both

their substrates (reactants) and their products than do chemical catalysts

Capacity for control: The catalytic activities of enzymes are subjected to regulations. The

mechanisms of regulations include allosteric control, covalent modification of enzymes, and

variation of the amounts of enzymes synthesized.

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In 1835, Jacob Berzelius pointed out that an extract of malt known as diastase (now known to contain

enzyme α-amylase) catalyzes the hydrolysis of starch. He developed the first general theory of chemical

catalysis. In 1878, Wilhelm Friedrich coined the name “enzyme” (Greek: en, in zyme, yeast) to mean

there is something in yeast, that catalyzes the reactions of fermentation. In 1897, Eduard Buchner

obtained a cell-free yeast extract that could carry out the synthesis of ethanol from glucose (alcoholic

fermentation). In 1894, Emil Fischer discovered that glycolytic enzymes can distinguish between

stereoisomeric sugars. He formulated the lock-and-key hypothesis. The lock-and-key hypothesis states

that the specificity of an enzyme (the lock) for its substrate (the key) arises from their geometrically

complementary shapes. According to the lock-and-key hypothesis, the substrate binding site may exist

in the absence of bound substrate. In 1926, James Sumner crystallized the first enzyme jack bean urease

demonstrating that these crystals are made up of proteins. It catalyzes the hydrolysis of urea to NH3 and

CO2. In 1963, the first amino acid sequence of an enzyme, bovine pancreatic ribonuclease A, was

reported in its entirety. In 1965, the first X-ray structure of an enzyme, hen egg white Lysozyme, was

elucidated.

Substrate specificity

Substrates are the reactants of enzyme catalyzed reactions. Enzymes are highly specific both in the

reactions that they catalyze and in their choice of substrates. Trypsin is quite specific and catalyzes the

splitting of peptide bonds only on the carboxyl side of lysine and arginine residues. Thrombin, an

enzyme that participates in blood clotting, is even more specific than trypsin. It catalyzes the hydrolysis

of Arg–Gly bonds in particular peptide sequences.

The specificity of an enzyme is due to the precise interaction of the enzyme with the substrate mediated

by multiple weak non-covalent interactions. The binding of substrate takes place at a particular site on

the enzyme called substrate-binding site. A substrate-binding site consists of an indentation or cleft on

the surface of an enzyme molecule that is complementary in shape (geometric complementarity), in size

(physical complementarity) and electrical charges (electronic complementarity) to the substrate. The

amino acid residues that form the substrate binding site are arranged to interact specifically with the

substrate in an attractive manner.

Stereospecificity

Enzymes involved with glucose metabolism are specific for D-glucose residues. Enzymes are highly

specific both in binding chiral substrates and in catalyzing their reactions. This stereospecificity stems

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from inherent chirality of amino acids and their ability to form asymmetric active sites. Yeast alcohol

dehydrogenase (YADH) catalyzes the interconversion of ethanol and acetaldehyde. Ethanol is a prochiral

molecule: isotope labeling experiment using deuterated ethanol showed that YADH can distinguish

between ethanol’s two methylene H atoms. If Pro s deuterium is used, none of the deuterium is

transferred from the product ethanol to NAD+ in the reverse reaction.

Geometric specificity

Most enzymes are quite selective about the identities of the chemical groups on their substrates.

Geometric specificity is a more stringent requirement than is stereospecificity.

Coenzymes

The standard set of twenty amino acids is suitable for catalyzing acid-base reactions and transient

covalent bond formations. However, the functional groups of enzymes are not suitable for catalyzing

oxidation–reduction and group-transfer reactions. The catalytic activity of these enzymes requires

Enzymes catalyze such reactions in the presence of small molecules termed cofactors, which function as

the enzymes’ “chemical teeth.” Cofactors can be of two major groups: (1) metals ions such as the Zn2+

are required for the catalytic activity of carboxypeptidase A and (2) small organic molecules such as the

NAD+ required for YADH are called coenzymes. Coenzymes like NAD+ are loosely and transiently

associated with a given enzyme molecule. In effect they are more like cosubstrates since they are

released from the enzyme after binding to it. Coenzymes are also chemically changed by the enzymatic

reactions in which they participate. The modified coenzyme must be returned to its original state by the

action of other enzymes.

The complete, catalytically active enzyme-cofactor complex is called a holoenzyme (Greek: holos, whole)

and inactive enzyme resulting from the removal of its cofactor is called an apoenzyme (Greek: apo,

away). Prosthetic groups are tightly bound coenzymes. Prosthetic groups are essentially permanently

associated with their proteins, often by covalent bonds. For example, the heme prosthetic group of

hemoglobin is tightly bound to its protein through extensive hydrophobic and hydrogen bonding

interactions together with a covalent bond between the heme Fe2+ ion and His.

Certain essential cofactors cannot be synthesized by some organisms and therefore these substances

must be acquired in diet. Many vitamins are coenzyme precursors. For example, nicotinamide

(alternatively known as niacinamide) or its carboxylic acid analog nicotinic acid (niacin) is a component

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of NAD+. The dietary deficiency disease of niacin in humans is known as pellagra. The dietary deficiency

disease of folic acid, which is precursor for tetrahydrofolate, is megaloblastic anemia. The dietary

deficiency disease of thymine B1, which is precursor for thymine pyrophosphate, is beriberi. The dietary

deficiency disease of cobalamine B12, which is precursor for 5’-deoxyadenosyl cobalamine, is pernicious

anemia.

Enzymes and coenzymes

Enzyme Coenzyme Reactions

Pyruvate dehydrogenase Thiamine pyrophosphate Aldehyde transfer

Monoamine oxidase Flavin adenine nucleotide Oxidation-reduction

Lactate dehydrogenase Nicotinamide adenine dinucleotid Oxidation-reduction

Glycogen phosphorylase Pyridoxal phosphate Amino group transfer

Acetyl CoA carboxylase Coenzyme A (CoA) Acyl transfer

Pyruvate carboxylase Biotin Carboxylation

Methylmalonyl mutase 5’-Deoxyadenosyl cobalamin

Thymidylate synthase Tetrahydrofolate One-carbon group

transfer

Cobalamin (B12) Alkylation

Lipoic acid Acyl transfer

Metal cofactors

Enzyme Cofactor

Carbonic anhydrase Zn2+

Carboxypeptidase Zn2+

EcoRV Mg2+

Hexokinase Mg2+

Urease Ni2+

Nitrogenase Mo

Glutathione peroxidase Se

Superoxide dismutase Mn

Acetyl CoA thiolase K+

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Enzyme regulation

A cell responds to changes in its environment, coordinates its numerous metabolic processes, and grows

and differentiates, all in an orderly manner.

Control of enzyme availability

The amount of a given enzyme in a cell depends on both its rate of synthesis and its rate of degradation.

E. coli cells grown in the absence of lactose lack the enzymes to metabolize this sugar. Within minutes of

exposure to lactose, however, these bacteria commence synthesizing the enzymes required to utilize

this nutrient. This is called induction. The degradation of proteins is also highly regulated.

Control of enzyme activity

The catalytic activity an enzyme can be directly controlled through the binding of small-molecule

effectors. These effectors change the enzyme’s substrate-binding affinity through conformational or

structural changes. Regulation of enzymes by allosteric effectors serves as a major control mechanism in

biological systems. The most studied exemplary proteins for allosteric regulations are hemoglobin and

aspartate transcarbamoylase (ATCase). The affinity of hemoglobin to oxygen is allosterically regulated by

the binding of ligands such as O2, CO2, H+, and BPG. These allostric effects result in cooperative

(sigmoidal) O2-binding curves. Similarly, allosteric changes alter ATCase’s substrate-binding sites.

Allosteric theory predicts activators preferentially bind to the enzyme’s active (R or high substrate

affinity) state, whereas inhibitors preferentially bind to the enzyme’s inactive (T or low substrate

affinity) state.

Aspartate transcarbamoylase (ATCase) catalyzes the formation of N-carbamoylaspartate from

carbamoyl phosphate and aspartate. This reaction is the first step unique to the biosynthesis of

pyrimidines. E. coli ATCase has the subunit composition c6r6, where c and r represent its catalytic and

regulatory subunits. The activity of the catalytic subunits is allosterically regulated by the regulatory

subunits. Homotropic and heterotropic effects are ligand bindings that alter the binding affinity of the

same or different ligands, respectively. ATCase exhibits positive homotropic cooperative binding to both

its substrates. Heterotropically, ATP, a purine nucleotide, is the activator whereas CTP, a pyrimidine

nucleotide, is the inhibitor. A common mode of metabolic control in which excess amounts of the

product a biosynthetic pathway controls the activity of an enzyme near the beginning of that pathway is

called feedback inhibition. Substrate binding induces a tertiary conformational shift in the catalytic

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subunits, which increases the subunit’s substrate-binding affinity and catalytic efficiency. This tertiary

shift is strongly coupled to large quaternary conformational shifts in the T and R states. Quaternary

changes, through which binding and catalytic effects are communicated among active sites, are

concerted or sequential. Allosteric regulations in other enzymes such as phosphofructokinase, fructose-

1,6-bisphosphatase, and glycogen phosphorylase operate in a similar manner.

Enzyme nomenclature

The International Union of Biochemistry and Molecular Biology (IUBMB) use a conventional, systematic

and functional classification and nomenclature of enzymes. Enzymes are classified and named according

to the nature of the chemical reactions they catalyze. There are six major classes of reactions that

enzymes catalyze. These are:

1. Oxidoreductases: oxidation–reduction reactions e.g. lactate dehydrogenase

2. Transferases: transfer of functional groups e.g. nucleoside monophosphate 9 kinase (NMP

kinase)

3. Hydrolases: hydrolysis reactions, or transfer of functional groups to water e.g. Chymotrypsin

4. Lyases: addition or elimination of groups to form double bonds e.g. fumarase

5. Isomerases: isomerization (intramolecular group transfer) e.g. triose phosphate isomerase

6. Ligases: ligation of two substrates through bond formation coupled with ATP hydrolysis e.g.

Aminoacyl-tRNA synthetase

Consequently, each enzyme is assigned two names and a four number classification. The accepted or

recommended name is an enzyme’s previously used name. It is a convenient name for everyday use.

Catalase is the accepted name for the enzyme that catalyzes the dismutation of H2O2 to H2O and O2. The

common name is not very informative. The systematic name involves appending the suffix -ase to the

names of their substrates specifying the type of reaction the enzyme catalyzes according to its major

group classification. The systematic name is used to minimize ambiguity.

For example:

Urease catalyzes the hydrolysis of urea

Alcohol dehydrogenase catalyzes the oxidation of alcohols to their corresponding aldehydes

ATP synthase is an enzyme that synthesizes ATP

A peptide hydrolase is an enzyme that hydrolyzes peptide bonds,

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The EC classification number can be obtained by considering all aspects of the enzyme catalyzed

reactions. For example, the systematic name for alcohol dehydrogenase is alcohol:NAD+ oxidoreductase

and the classification number is EC 1.1.1.1. Lysozyme is a common name. Its systematic name is

peptidoglycan N-acetylmuramoylhydrolase. The classification number is EC 3.2.1.17. The first number

(3) indicates the enzyme’s major class (hydrolases), the second number (2) denotes its subclass

(glycosylases), the third number (1) designates its sub-subclass (enzymes hydrolyzing O- and S-glycosyl

compounds), and the fourth number (17) is the enzyme’s arbitrarily assigned serial number in its sub-

subclass. The designation for nucleoside monophosphate (NMP) kinase is EC 2.7.4.4. The first number

(2) indicates the enzyme’s major class (transferase) and the second number (7) denotes its subclass

(phosphoryl group transferases). Various functional groups can accept the phosphoryl group. The third

number (4) designates the phosphate group acceptor. In regard to NMP kinase, a nucleoside

monophosphate is the acceptor. The final number (4) is the arbitrarily assigned serial number for NMP.

Rates of enzymatic reactions

Kinetics is the study of reaction rates. Reactions that are directly proportional to the reactant

concentration are first-order reactions whereas bimolecular reactions have second-order rate constants.

The goal of kinetic theory is to describe reaction rates in terms of the physical properties of the reacting

molecules. A reaction can be zero order when the rate is independent of reactant concentrations.

Enzyme-catalyzed reactions can approximate zero-order reactions when substrate concentration is far

greater than enzyme concentration. Enzyme kinetics studies the rate of an enzyme-catalyzed reaction

and its variation with the reaction conditions. A reaction mechanism is a detailed description of the

various steps in a chemical reaction and the sequence with which they occur. Kinetic measurements of

enzyme-catalyzed reactions are among the most powerful techniques for elucidating the catalytic

mechanisms of enzymes. The major objectives of enzyme kinetics are

to determine the binding affinities of substrates and inhibitors to an enzyme and the maximum

catalytic rate of an enzyme

to understand the role of an enzyme in an overall metabolic process

to apply measurements of enzyme-catalyzed reactions for biochemical and clinical analyses

to elucidate the enzyme’s catalytic mechanism by combining kinetic data with chemical and

structural studies

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Kinetic measurements can provide a phenomenological description of enzymatic behavior, but are

incapable of distinguishing the number of intermediates. Intermediates must be detected and

characterized by independent means such as by spectroscopic analysis. Kinetic data cannot

unambiguously establish a reaction mechanism. A postulated mechanism can be very simple, elegant,

and rational and fully accounts for the observed kinetic data, but it is just one of the infinite numbers of

alternative mechanisms. Conversely, a mechanism incompatible with a given kinetic data must be

rejected.

The transition state theory

The transition state theory or absolute rate theory was developed in the 1930s, principally by Henry

Eyring. It predicts the existence of a high-energy (unstable) complex in which transient covalent bonds

are in the process of forming and breaking. The transition state is a transitory molecular structure that is

no longer the substrate but is not yet the product. Once the reaction enters the transition state, it

collapses to either substrate or product with equal probability, but which of the two accumulates is

determined only by the Gibbs free energy difference. Transition state theory assumes that the transition

state attains rapid equilibrium with the reactants. The formation of the activated complex is postulated

to be the rate-determining step of a reaction since its concentration is small.

Thermodynamics of the transition state

A chemical reaction of reactants (R) goes through a transition state (X‡) to form products (P). The

minimum free energy pathway of a reaction is known as its reaction coordinate. The reaction coordinate

diagram or a transition state diagram shows the free energy of the reactants and products along the

reaction coordinate. The transition state or activated complex is the highest-energy species in a reaction

coordinate. Transition state is the least-stable reaction intermediate which is too unstable to exist for

long. It is only metastable (like a ball balanced on a pin). Consequently, it is seldom-occupied species

along the reaction pathway. Enzymes catalyze reactions by stabilizing the transition states. The

mechanism of enzyme catalysis can be understood in terms of two thermodynamic properties of the

reaction. These are the Gibbs free-energy difference (ΔG) and the activation energy (ΔG‡).

Gibbs free energy (G)

Gibbs free energy (G) is a thermodynamic property that is a measure of useful energy of a system.

Useful energy is the energy that is capable of doing work. The standard free-energy change of a reaction

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is related to the equilibrium constant. The position of equilibrium is a function only of the Gibbs free-

energy difference (ΔG) between reactants and products. It depends only on the free energy of the

products (the final state) minus the free energy of the reactants (the initial state) and determines

whether the reaction will take place spontaneously.

1. A reaction can take place spontaneously only if ΔG is negative. Such reactions are said to be

exergonic.

2. A system is at equilibrium and no net change can take place if ΔG is zero.

3. A reaction cannot take place spontaneously if ΔG is positive. An input of free energy is required

to drive such a reaction. These reactions are termed endergonic.

The Gibbs free energy of activation (ΔG‡)

The Gibbs free energy of activation or simply activation energy is the minimum amount of energy

required to initiate the conversion of reactants into products. It is the difference in free energy between

the transition state and the substrate. The rate constant for passage of the activated complex over the

energy maximum is called activation barrier or the kinetic barrier of the reaction. The rate of a reaction

decreases exponentially with ΔG‡. The larger the difference between the free energy of the transition

state and that of the reactants, that is, the less stable the transition state, the slower the reaction

proceeds. An increase in thermal energy drives the reacting complex over the activation barrier and that

is how rising temperature speeds up a reaction.

Enzymes accelerate reactions by facilitating the formation of the transition state. Enzymes alter the

reaction rate by lowering activation energy. When the activation energy is lowered, more and more

molecules will have the minimum energy required to reach the transition state. Enzymes facilitate the

formation of the transition state. Enzymes equally accelerate the forward and the reverse reactions so

that the equilibrium constant for the reaction remains unchanged. Linus Pauling wrote “I think that

enzymes are molecules that are complementary in structure to the activated complexes of the reactions

that they catalyze; that is, to the molecular configuration that is intermediate between the reacting

substances and the products of reaction for these catalyzed processes. The attraction of the enzyme

molecule for the activated complex would thus lead to a decrease in its energy and hence to a decrease

in the energy of activation of the reaction and to an increase in the rate of reaction.”

Active sites of enzymes

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The first step in enzymatic catalysis is the formation of an enzyme–substrate (ES) complex. The active

site of an enzyme is a particular site on the enzyme that binds to the substrates (and a cofactor, if any).

The active site is shaped like a three-dimensional cleft, or crevice to which the substrates bind. Active

sites are unique microenvironments in which water is completely excluded unless it is a reactant. Amino

acid residues in the active site participate directly in the making and breaking of bonds. These residues

are called the catalytic groups. The active site takes up only a small part of the total volume of an

enzyme.

Evidences for the formation of discrete enzyme–substrate (ES) complex come from the fact that

enzymes are highly selective toward their substrates. Besides, all enzyme-catalyzed reactions have

maximal velocities suggesting saturation effect. Moreover, the spectroscopic characteristics of many

colored enzymes (with prosthetic groups) and colored substrates change upon the formation of an ES

complex. Finally, X-ray crystallography has provided high-resolution images of substrates and substrate

analogs bound to the active sites of many enzymes.

The binding energy is the free energy released during the formation of a large number of weak

interactions between an enzyme and its complementary substrate. The binding energy between enzyme

and substrate is important for catalysis. The full complement of such interactions is formed only when

the substrate is converted into the transition state. Therefore, enzymes bind to the transition state of

the catalyzed reaction in preference to the substrate. The energy required to generate the transition

state is released when the transition state forms the product. Transition states bind to the active sites of

enzymes by reversible, multiple weak non-covalent interactions: electrostatic interactions, hydrogen

bonds, and van der Waals forces. These are attractive forces on the enzyme that lure the substrate to

the active site. These forces are poetically known as Circe effects.

Models for enzyme-substrate interactions

The lock-and-key model of enzyme-substrate binding

In 1890, Emil Fischer proposed the lock-and-key model of enzyme–substrate binding. In this model, the

active site of the unbound enzyme is complementary in shape to the substrate in analogy with lock and

key. The enzyme and its substrate should have complementary shapes like lock and key. The directional

character of hydrogen bonds between enzyme and substrate often enforces a high degree of specificity.

The specificity of binding depends on the precisely defined arrangement of atoms in an active site.

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Induced-fit model of enzyme-substrate binding

In this model, the enzyme changes its shape on substrate binding. The active site forms a shape

complementary to the substrate only after substrate binding. Induced-fit is a process of dynamic

recognition in which the binding energy promotes structural changes in both the enzyme and the

substrate. Enzymes are flexible and that the shapes of the active sites can be markedly modified by the

binding of substrate. Conformation selection is a process in which a substrate can bind to only certain

conformations of an enzyme. The mechanism of catalysis is dynamic, involving structural changes with

multiple intermediates of both substrates and the enzyme generating the optimal alignment of

functional groups at the active site so that maximum binding energy occurs only between the enzyme

and the transition state. X-ray studies indicate that the substrate-binding sites of most enzymes are

largely preformed in accordance with lock-and-key model but most of them also exhibit at least some

degree of induced- fit upon binding substrate.

Multistep reactions have rate-determining steps

The rate-determining step of an overall multistep reaction is the one is much slower than the others.

The slow step acts as a “bottleneck” and it has the highest activation barrier in the chain. The rate of

formation of product P can only be as fast as the slowest elementary reaction.

The Michaelis–Menten model of enzyme kinetics

Hydrolysis of sucrose is catalyzed by the yeast enzyme invertase (α-fructofuranosidase).

Sucrose + H2O→ glucose + fructose

When the concentration of sucrose is high enough to saturate the enzyme, the reaction rate becomes

independent of the sucrose concentration; that is, the rate is zero order with respect to sucrose. The

overall reaction is composed of two elementary reactions in which the substrate forms a complex with

the enzyme that subsequently decomposes to products and enzyme.

E + S→ ES→ P + E Where E, S, ES, and P symbolize the enzyme, substrate, enzyme–substrate complex,

and product, respectively. The high substrate concentration converts the entire enzyme populations to

the ES form. The second step of the reaction becomes rate limiting and the overall reaction rate is

insensitive to further addition of substrates. The overall velocity (rate) of this reaction is the difference

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between the rates of the elementary reactions leading to the appearance of the complex and those

resulting in its disappearance.

Two assumptions were introduced to simplify and facilitate the description enzyme kinetics. These were

assumption of equilibrium and assumption of steady state.

Assumption of equilibrium: In 1913, Leonor Michaelis and Maud Menten proposed that k–1 ≫ k2 so as to

bring the first step of the reaction into equilibrium. They also proposed the rate of product formation is

negligible and thus no back reaction (k-2=0). The non-covalently bound enzyme–substrate complex ES is

also known as the Michaelis complex and its dissociation constant is the reciprocal of the equilibrium

constant. The critical feature in this assumption is that a specific ES complex is a necessary intermediate

during catalysis.

Assumption of steady state: In 1925, George E. Briggs and John B.S. Haldane first proposed the steady-

state assumption. An enzyme catalyzed reaction can be divided into two phases: the transient phase and

the steady-state phase. The transient phase is the initial stage of the reaction at reaction times close to

zero when the reverse reaction is insignificant. It is usually over within milliseconds of mixing the

enzyme and substrate. The transient phase is followed by the steady state phase in which concentration

of enzyme-substrate complex stay the same even if the concentrations the substrates and products are

changing. The ts denotes the time when the steady state is first achieved. The steady state is maintained

over most of the course of the reaction until the substrate is nearly exhausted. The rate of synthesis or

formation of ES equals its rate of consumption or breakdown.

Initial velocity (VO)

The initial rate of catalysis or initial velocity (VO) is the number of moles of the product formed per

second when the reaction is just beginning i.e. in the transient phase. Initial velocity is operationally

defined as the velocity measured before more than 10% of the substrate has been converted into

products. The plot of the concentration of substrates consumed or products formed as a function of

time is called progress curve. The slope of the progress curve at the transient phase of a reaction is the

initial velocity for each substrate concentration. The use of initial velocity minimizes complicating factors

such as the effects of reversible reactions, inhibition of the enzyme byproducts, and progressive

inactivation of the enzyme.

Michaelis–Menten equation

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Michaelis–Menten equation is a kinetic expression of enzyme-catalyzed reactions in terms of

experimentally measurable quantities: the total enzyme concentration ([E]T) and initial substrate

concentration ([S]). The plot of the initial velocity (VO) or amount of product formed versus the substrate

concentration [S], assuming a constant amount of enzyme is rectangular hyperbola.

([ ]

[ ] )

The rate of catalysis rises as substrate concentration increases and then begins to level off and approach

a maximum at higher substrate concentrations. At very low substrate concentration, the reaction is first

order with the rate directly proportional to the substrate concentration. At high substrate concentration

([S] ≫ KM), the reaction is zero order with rate independent of substrate concentration.

Limitations

Allosteric enzymes, containing multiple subunits and multiple active sites, often display sigmoidal plots

rather than the hyperbolic plots predicted by the Michaelis–Menten equation. The Michaelis–Menten

model implicitly neglects enzymatic reverse reactions. Yet many enzymatic reactions are highly

reversible.

Lineweaver–Burk or double-reciprocal plot

The parameters of the Michaelis–Menten equation, Vmax and KM, are very important characteristics of an

enzyme. They are usually determined experimentally from kinetic data. KM is equal to the substrate

concentration that yields Vmax/2. At very high values of [S], the initial velocity Vo asymptotically

approaches Vmax. However, Vmax can only be approached but never attained. A simple method for

determining the values of Vmax and KM was formulated by Hans Lineweaver and Dean Burk by taking the

reciprocal of MM Equation. The Lineweaver–Burk or double-reciprocal plot is a linear plot of 1/vo versus

1/[S]. For this straight line, the y- intercept is 1/Vmax, the extrapolated x-intercept is -1/KM and the slope

is KM/Vmax.

Limitations

Most experimental measurements involve relatively high [S] and are hence the graph is crowded onto

the left side. Besides, for small values of [S], small errors in Vo lead to large errors in 1/Vo and resulting

in large errors for the calculated KM and Vmax.

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Maximum velocity (Vmax)

The maximal velocity of a reaction, Vmax, is attained at high substrate concentration when the catalytic

sites on the enzyme are saturated with substrate. The total enzyme concentration [E]T is the sum of

concentration of uncombined enzyme [E] and the concentration of the [ES] complex. The velocity is

maximal when the enzyme is entirely in the ES form.

Michaelis constant (KM)

Michaelis constant (KM) is the substrate concentration at which the reaction rate is half-maximal or half

the active sites of enzymes are filled. It is a useful characteristic of enzyme–substrate interactions. KM is

a measure of the substrate concentration required to achieve significant rate of catalysis. Most people

have two forms of the alcohol dehydrogenase in the liver, a low KM mitochondrial form and a high KM

cytoplasmic form. Alcohol dehydrogenase converts ethanol into acetaldehyde. The magnitude of KM

varies widely with the identity of the enzyme, the nature of the substrate and environmental conditions

such as pH, temperature, and ionic strength. Under circumstances when k–1 ≫ k2, KM approaches KD, the

dissociation constant of the Michaelis complex and it reflects the strength of the enzyme–substrate

interaction. As KD decreases, the affinity of the enzyme for its substrate increases indicating strong

binding. The KM values of many enzymes approximate the in vivo concentrations of their substrates

suggesting that most enzymes evolved to have a proactive KM value. Under this situation, the enzyme

will display significant activity and yet the activity will be sensitive to changes in substrate concentration.

Catalytic efficiency

Maximal velocity is related to the catalytic rate constant of an enzyme k2 (kcat) also known as the

turnover number of an enzyme. The turnover number of an enzyme (kcat) is the number of substrate

molecules converted into product by an enzyme molecule per unit time when the enzyme is fully

saturated with substrate. Under the proper conditions (KM ≫[S]), the enzyme-catalyzed reaction is a

second order with the apparent second-order rate constant kcat/KM. The rate of an enzyme-catalyzed

reaction is directly proportional to the concentration of its enzyme–substrate complex, which, in turn,

varies with the enzyme and substrate concentrations and with the enzyme’s substrate-binding affinity.

The rate varies directly with how often enzyme and substrate encounter one another in solution. The

quantity kcat/KM is the measure of an enzyme’s catalytic efficiency.

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The ultimate limit on the value of kcat/KM is set by k1, the rate of formation of the ES complex. This rate

cannot be faster than the diffusion-controlled encounter of an enzyme and its substrate. Hence, there is

a maximum limit for kcat/KM. Many enzymes such as catalase, superoxide dismutase, fumarase, carbonic

anhydrase, acetylcholinesterase, and triose phosphate isomerase have attained virtual catalytic

perfection. Every encounter between enzyme and substrate is productive. Enzymes catalyzing

subsequent reactions are organized into complexes so that the product of one enzyme can be rapidly

found by the next enzyme. The products are channeled from one enzyme to the next, much as in an

assembly line. SOD superoxide dismutase (SOD) is responsible to inactivate the highly reactive and very

destructive superoxide radical. It binds both a Cu2+ and a Zn2+ ion. The arrangement of charged groups

on the enzyme’s surface appears to guide the charged substrate electrostatically to the enzyme’s active

site. Other enzymes have similar mechanisms to funnel polar substrates to their active sites.

The Hill equation

In allosteric enzymes, the binding of a substrate to one active site can alter the properties of other active

sites in the same enzyme molecule through cooperative effects. Besides, the activity of an allosteric

enzyme can be altered by regulatory molecules that reversibly bind to specific sites other than the

catalytic sites. Such cooperative binding results in a sigmoidal plot of initial velocity versus substrate

concentration. The Hill equation is a kinetic expression of a reversible enzyme-catalyzed reaction in

which substrate binding sites are not independent of each other. It describes the degree of saturation of

a multisubunit protein as a function of ligand concentration. The fractional saturation function for

oxygen binding to hemoglobin has the same functional form as Hill equation.

Inhibition

Inhibitors are specific substances that reduce the catalytic efficiency of enzymes. Enzyme inhibitors are

either irreversible inhibitors or reversible inhibitors. Inhibitors are commonly used to probe the chemical

and conformational nature of a substrate-binding site in an effort to elucidate the enzyme’s catalytic

mechanism. Many enzyme inhibitors are drugs.

Irreversible inhibitors

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An irreversible inhibitor is tightly bound to its target enzyme, either covalently or non-covalently and

dissociates very slowly. An irreversible inhibitor is also known as inactivator, as is any agent that

somehow inactivates the enzyme. Inactivators truly reduce the effective level of [E]T at all values of [S].

Some important drugs are irreversible inhibitors. Aspirin acts by covalently modifying the enzyme

cyclooxygenase, thereby reducing the synthesis of inflammatory signaling molecules. Penicillin acts by

covalently modifying the enzyme transpeptidase, thereby preventing the synthesis of bacterial cell walls.

Bacterial cell wall is made up of a peptidoglycan which consists of linear polysaccharide chains that are

cross-linked by short peptides (pentaglycines and tetrapeptides). Glycopeptide transpeptidase catalyzes

the formation of the cross-links that make the peptidoglycan so stable. Penicillin irreversibly inhibits the

cross-linking transpeptidase by the Trojan horse stratagem.

Suicide inhibitors

Irreversible inhibitors can be used to determine the functional groups required for enzyme activity and

map the active sites of enzymes. Suicide inhibitors, or mechanism-based inhibitors, are modified

substrates that specifically modify an enzyme’s active site. They are also known as affinity labels, group

specific reagents or reactive substrate analogs. Affinity labels are molecules that are structurally similar

to enzyme substrate and are able to covalently bind to active-site residues. These group-specific

reagents can react selectively with specific side chains of amino acids. Diisopropylphosphofluoridate

(DIPF) is an irreversible inhibitor of the proteolytic enzyme chymotrypsin. It modifies only 1 of the 28

serine residues suggesting that the labeled serine residue is especially reactive. Tosyl-L-phenylalanine

chloromethyl ketone (TPCK) is a substrate analog inhibitor of chymotrypsin. It reacts irreversibly with a

histidine residue at the active site. 3-bromoacetol phosphate is an irreversible inhibitor for the enzyme

triose phosphate isomerase (TPI). It mimics the normal substrate, dihydroxyacetone phosphate, and

covalently modifies the enzyme at the active site. The mechanism of catalysis then generates a

chemically reactive intermediate that inactivates the enzyme through covalent modification. N, N-

dimethylpropargylamine is an inhibitor of the enzyme monoamine oxidase (MAO). It is oxidized by the

flavin prosthetic group of monoamine oxidase which in turn inactivates the enzyme by binding to N-5 of

the flavin prosthetic group.

Reversible inhibitors

Reversible inhibitors are characterized by a rapid dissociation of the enzyme–inhibitor complex. The

most convenient means of monitoring reversible enzyme inhibition is plotting the initial velocity vo of a

17 | P a g e

simple Michaelis–Menten reaction versus the substrate concentration [S] in the presence of different

concentrations inhibitors. Reversible inhibition can be grouped into competitive, non-competitive and

uncompetitive inhibition.

Competitive inhibition

In competitive inhibition, an enzyme can bind to either a substrate (forming an ES complex) or an

inhibitor (EI) but not to both (ESI, enzyme–substrate–inhibitor complex). A competitive inhibitor binds at

the active site preventing substrate binding. The general model for competitive inhibition is given by a

reaction scheme showing the formation of ES, and EI. The enzyme–inhibitor complex is catalytically

inactive. Many competitive inhibitors are substances that structurally resemble their enzyme’s substrate

but they either do not react or react very slowly compared to the substrate. The inhibitor competes

directly with the normal substrate for the same active (binding) site on the enzyme. There is true

competition between I and S for the enzyme’s substrate-binding site; their binding is mutually exclusive.

A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules

that are bound to substrate. Competitive inhibition can be relieved by sufficiently high concentrations of

substrate.

Succinate dehydrogenase, that converts succinate to fumarate, is competitively inhibited by malonate.

Many competitive enzyme inhibitors are effective drugs. Methotrexate (also called amethopterin), a

structural analog of dihydrofolate, is a potent competitive inhibitor dihydrofolate reductase. It binds

tightly to the enzyme dihydrofolate reductase, thereby preventing it from carrying out the reduction of

dihydrofolate to tetrahydrofolate. Tetrahydrofolate is essential cofactor in the biosynthesis of the DNA

precursor dTMP. Methotrexate is very active in rapidly dividing cells such as cancer cells. Statins are

drugs that reduce high cholesterol levels by competitively inhibiting a key enzyme in cholesterol

biosynthesis. Drugs such as ibuprofen are competitive inhibitors of enzymes that participate in signaling

pathways in the inflammatory response.

Transition-state analogs

The theory that enzymes bind to transition-states with higher affinity than substrates has led rational

drug-designing based on specific mechanism of an enzyme-catalyzed reaction. Compounds resembling

the transition-state, transition-state analogs, are potent competitive inhibitors. The inhibitory power of

transition-state analogs underscores the essence of catalysis: selective binding of the transition state.

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Proline racemase, which catalyzes the racemization of proline, is competitively inhibited by the planar

analogs of proline, pyrrole-2-carboxylate and Δ-1-pyrroline- 2-carboxylate.

Transition-state analogs are sources of insight into the mechanism of enzyme action: specific inhibitors

can often be used to identify residues critical for catalysis, can serve as potent and specific competitive

inhibitors of enzymes, and are used as immunogens to generate a wide range of novel catalysts.

Ferrochelatase, the final enzyme in the biosynthetic pathway for the production of heme, catalyzes the

insertion of Fe2+ into protoporphyrin IX. The nearly planar porphyrin must be bent for iron to enter. N-

Methylmesoporphyrin is a transition-state analog used as antigen to generate catalytic antibodies

(abzymes). Abzymes catalyzing many kinds of chemical reactions have been produced by using similar

strategies.

Kinetics of a competitive inhibitor

The different types of reversible inhibitors are kinetically distinguishable. A competitive inhibitor acts by

reducing the concentration of free enzyme available for substrate binding. The presence of competitive

inhibitor has the effect of making [S] appears more dilute than it actually is, or alternatively, making KM

appears larger than it really is. In effect, a competitive inhibitor increases the apparent value of KM. As

the concentration of a competitive inhibitor increases, higher concentrations of substrate are required

to attain a particular reaction velocity. The quantities KappM and Vappmax are the “apparent” values of KM

and Vmax of the Michaelis–Menten equation that would actually be observed in the presence of inhibitor.

A competitive inhibitor has no effect on the turnover number of the enzyme. In the presence of a

competitive inhibitor, an enzyme will have the same Vmax as in its absence. At a sufficiently high

concentration, virtually all the active sites are filled with substrate. The hallmark of competitive

inhibition in double-reciprocal plots is the intersection of the plots at various concentrations of I at

1/Vmax on the 1/vo axis (constant Vmax). The dissociation constant for the inhibitor is given by the

following equation.

[ ][ ]

[ ]

The smaller the Ki, the more potent the inhibition is.

Uncompetitive inhibition

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In uncompetitive inhibition, the binding site for the inhibitor is created only on interaction of the

enzyme and its substrate. Uncompetitive inhibition is essentially substrate-dependent inhibition in that

the inhibitor binds only to the enzyme–substrate complex but not to the free enzyme. Both the inhibitor

and substrate can bind simultaneously to an enzyme molecule at different binding sites. The binding of

the uncompetitive inhibitor might cause structural distortion of the active site, thereby rendering the

enzyme catalytically inactive. The general model for competitive inhibition is given by a reaction scheme

showing the formation of ES and ESI complex but ESI complex does not go on to form any product. The

uncompetitive inhibitor depletes ES. To maintain the equilibrium between E and ES, more S binds to E,

increasing the apparent value of k1 and lowering the apparent value of KM. Since there will always be

some unproductive ESI complex, Vappmax will be reduced. Uncompetitive inhibition exhibits the opposite

behavior of a competitive inhibitor in that the effect of an uncompetitive inhibitor is negligible at low

substrate concentration and it cannot be relieved by the addition of more substrate. Glyphosate, also

known as Roundup, is an important class of herbicides that functions as uncompetitive inhibitor of the

enzyme in the biosynthetic pathway for aromatic amino acids.

Kinetics of an uncompetitive inhibition

In uncompetitive inhibition, Vmax cannot be attained even at high substrate concentrations. The

apparent value for KM is lowered, becoming smaller as more inhibitor is added. The diagnostic feature of

uncompetitive inhibition in the Lineweaver–Burk plots at various uncompetitive inhibitor concentrations

is a constant slope producing parallel lines. Uncompetitive inhibitor affects the catalytic function of the

enzyme but not its substrate binding. The dissociation constant for the inhibitor is given by the following

equation.

[ ][ ]

[ ]

Noncompetitive inhibition

In noncompetitive inhibition, the inhibitor binds both to the free enzyme and to an enzyme–substrate

complex. A noncompetitive inhibitor binds to enzyme sites that participate in both substrate binding and

catalysis. This type of inhibitor both hinders the binding of substrate and decreases the turnover number

of the enzyme. A noncompetitive inhibitor acts by diminishing the concentration of functional enzyme.

Noncompetitive inhibition is alternatively known as mixed inhibition. A mixed inhibitor is effective at

both high and low substrate concentrations.

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Kinetics of a noncompetitive inhibitor

In noncompetitive inhibition, KM remains unchanged and the value of Vmax is decreased. The net effect is

to decrease the turnover number. Noncompetitive inhibition cannot be overcome by increasing the

substrate concentration. In pure noncompetitive inhibition, the dissociation constant Ki for the inhibitor

binding to E is the same as for binding to ES complex. Noncompetitive inhibition cannot be overcome by

increasing the substrate concentration. Doxycycline, an antibiotic, functions at low concentrations as a

noncompetitive inhibitor of a proteolytic enzyme (collagenase). It is used to treat periodontal disease.

Some of the toxic effects of lead poisoning may be due to lead’s ability to act as a noncompetitive

inhibitor of a host of enzymes. Lead reacts with crucial sulfhydryl groups in these enzymes.

Effects of pH

The initial rates for many enzymatic reactions exhibit bell-shaped curves as a function of pH. These

curves reflect the requirement for amino acid residues to be in a specific ionization state for enzyme

activity. Most proteins are active only within a narrow pH range called optimal range (typically 5 to 9).

The ionization constants of enzymes can be evaluated by the analysis of the curves of log Vmax versus pH.

The measured pK’s often provide valuable clues as to the identities of the amino acid residues essential

for enzymatic activity. For example, a measured pK of 4 suggests that an Asp or Glu residue is essential

to the enzyme. Similarly, pK’s of 6 or 10 suggest the participation of a His or a Lys residue, respectively.

The identification of a kinetically characterized pK with a particular amino acid residue must be verified

by other types of measurements such as the use of group-specific reagents to inactivate a putative

essential residue Mechanistic conclusions based on kinetic analyses alone are fraught with uncertainties

and are easily confounded by inaccurate experimental data. The pH of the medium affects:

the binding of substrate to enzyme,

the catalytic activity of the enzyme,

the ionization of substrate, and

the variation of protein structure (usually significant only at extremes of pH)

Catalytic mechanisms

Enzymes are not passive surfaces on which reactions take place but, rather, are complex molecular

machines that operate through a great diversity of mechanisms. Catalysis is a process that increases the

rate at which a reaction approaches equilibrium. Catalysts stabilize the transition state with respect to

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the un-catalyzed reaction. The most striking characteristics of enzymes are their catalytic power and

specificity but what are the sources of the catalytic power and specificity of enzymes? Enzymes are

powerful catalysts due to two related properties: their specificity of substrate binding combined with

their optimal arrangement of catalytic groups. The catalytic mechanisms of enzymes can be revealed

through the use of experimental probes, techniques of protein structure determination and site-

directed mutagenesis. The types of catalytic mechanisms that enzymes employ have been classified as:

1. Acid–base catalysis.

2. Covalent catalysis.

3. Metal ion catalysis.

4. Electrostatic catalysis.

5. Approximation catalysis.

6. Preferential binding of the transition state complex

Acid–base catalysis

General acid catalysis is a process in which partial proton transfer from a Brønsted acid (a species that

can donate protons) lowers the free energy of a reaction’s transition state. Many biochemically

important reactions are susceptible to acid and/or base catalysis. These include the hydrolysis of

peptides, nucleic acids and esters, the reactions of phosphate groups, tautomerizations, and additions to

carbonyl groups. The ability of enzymes to arrange several catalytic groups about their substrates makes

concerted acid–base catalysis a common enzymatic mechanism. The side chains of the amino acid

residues Asp, Glu, His, Cys, Tyr, and Lys have pK’s in or near the physiological pH range to enable

enzymes act as general acid and/or base catalysts.

Bovine pancreatic ribonuclease A (RNase A)

RNase A is digestive enzyme that hydrolyzes RNA to its component nucleotides. The reaction exhibits a

pH profile that peaks near pH 6. This pH profile together with chemical derivatization and X-ray

structural studies indicates that RNase A has two essential His residues, His-12 and His-119, which act in

a concerted manner as general acid and base catalysts.

Covalent catalysis

Covalent catalysis is a process in which the rate of a reaction is accelerated through the transient

formation of a catalyst–substrate covalent bond. Covalent catalysis requires a nucelophilic, electrophilic

22 | P a g e

and elimination stages. The nucleophilic stage is a reaction between the catalyst and the substrate to

form a covalent bond. The electrophilic stage is the withdrawal of electrons from the reaction center by

the now electrophilic catalyst. Elimination of the catalyst is essentially the reverse of the nucleophilic

stage. The proteolytic enzyme chymotrypsin and the decarboxylation of acetoacetate provide excellent

example of covalent catalysis. In the first stage of this reaction, the primary amine nucleophilically

attacks the carbonyl group of acetoacetate to form a Schiff base (imine bond). The active site contains a

reactive group, usually a powerful nucleophile, which becomes temporarily covalently attached to a part

of the substrate in the course of catalysis. The side chains of the amino acid residues with high

polarizabilities (highly mobile electrons), such as imidazole and thiol as well as coenzymes make good

covalent catalysts. Other enzyme functional groups that participate in covalent catalysis include the

carboxyl function of Asp, and the hydroxyl group of Ser. In addition, several coenzymes such as thiamine

pyrophosphate and pyridoxal phosphate function as covalent catalysts.

Metal ion catalysis

Metal ion catalysis is a process in which metal ions participate in the catalytic process in three major

ways. These are

1. The metal ion facilitates the formation of nucleophiles such as hydroxide ion by direct

coordination. It mediates oxidation–reduction reactions through reversible changes in the metal

ion’s oxidation state. E.g. Zn2+ in carbonic anhydrase.

2. The metal ion can serve as an electrophile, electrostatically stabilizing or shielding negative

charges on a reaction intermediate. E.g. Mg2+ ion in EcoRV.

3. The metal ion may serve as a bridge between enzyme and substrate, increasing the binding

energy and holding the substrate in a conformation appropriate for catalysis. It binds to

substrates so as to orient them properly for reaction. E.g. For myosin, a phosphate group of the

ATP substrate serves as a base to promote its own hydrolysis. Mg2+ ion functions as a bridge in

myosin and all enzymes that utilize ATP as a substrate.

Nearly one-third of all known enzymes require the presence of metal ions for catalytic activity. Metal ion

catalysis can be carried out by metalloenzymes and metal-activated enzymes. Metalloenzymes contain

tightly bound transition metal ions such as Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, or Co3+. Metal-activated enzymes

contain loosely bound alkali and alkaline earth metal ions such as Na+, K+, Mg2+, or Ca2+. Metal ions are

far more effective catalysts than protons because metal ions can be present in high concentrations at

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neutral pH and can have charges greater than 1. Consequently, metal ions have been dubbed

“superacids”. The charge on metal ions makes its bound water molecules more acidic than free H2O and

therefore a source of OH- ions even below neutral pH. Metal ion–bound hydroxyl group is a potent

nucleophile. Carbonic anhydrase catalyzes the hydration of carbon dioxide which is useful for the

transfer of CO2 from the tissues to the blood and then to the air in the alveolae. It contains an essential

Zn2+ ion tetrahedrally coordinated by three evolutionarily invariant His side chains. One of the conserved

histidine residues facilitates the removal of a hydrogen ion from a zinc-bound water molecule to

generate hydroxide ion.

Electrostatic catalysis

Electrostatic catalysis is a process in which the charge distributions about the active sites of enzymes

1. are arranged to stabilize the transition states

2. guides polar substrates toward their binding sites

Substrate binding is accompanied by the exclusion of water from an enzyme’s active site. Therefore, the

local dielectric constant of the active site resembles that in an organic solvent, where electrostatic

interactions are much stronger than they are in aqueous solutions. The rates of some enzymatic

reactions are greater than their apparent diffusion-controlled limits due to substrate-guiding effects.

Catalysis by approximation

Catalysis by approximation is a process in which enzymes accelerate the reaction by properly orienting

substrates and arresting their relative motions. Catalysis by approximation is catalysis through

orientation, proximity and arresting effects. Enzymes bind substrates in a manner that both aligns and

immobilizes them so as to optimize their reactivities. The free energy required for alignment and

immobilization is derived from the specific binding free energy of substrate to enzyme. A substrate may

be maximally reactive only when it assumes a conformation that aligns its various orbitals in a way that

minimizes the electronic energy of its transition state. The minimum electronic energy of the transition

state that maximizes its reactivity is called stereoelectronic control. This is called catalysis through

orientation effects.

Many reactions have two distinct substrates. These substrates react most readily when they have the

proper spatial relationship. The reaction rate can be considerably enhanced by bringing the two

substrates together along a single binding surface on an enzyme. This is catalysis through proximity

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effects. For example, carbonic anhydrase binds carbon dioxide and water in adjacent sites to facilitate

their reaction. The rate of an intramolecular reaction can be greatly increased by arresting a molecule’s

internal motions in a way that increases the mole fraction of the reacting groups that are in a

conformation which can enter the transition state. An enzyme freezes out the relative translational and

rotational motions of its substrates, thereby decreasing their entropy, to enhance their reactivity. This is

catalysis through arresting effects.

Catalysis by preferential transition state binding

The binding of an enzyme to the transition state with greater affinity than the corresponding substrates

or products is the most important mechanisms of enzymatic catalysis. Interactions that preferentially

bind the transition state increase its concentration and proportionally increase the reaction rate. An

excellent substrate does not necessarily bind to its enzyme with high affinity, but does so, on activation

to the transition state.

Many-molecules experiments

Many-molecules experiments, also known as ensemble studies, are designed to investigate the average

characteristic properties of a presumably uniform collection (ensemble) of molecules. The signal

obtained when recording many molecules at the same time represents the average property of the bulk.

The basic assumption of many-molecules experiments is that all of the molecules of an ensemble are the

same or very similar. Currently, there are many well established many-molecules experiments including

spectroscopic and crystallographic techniques. For a perfectly uniform ensemble, the property of a

single molecule is the same as the average property of the bulk. However, molecular heterogeneity, the

ability of a molecule, over time, to assume several different structures that differ slightly in stability, is

an inherent property of all large biomolecules. Biomolecules might not be perfect ensembles

(homogeneous populations) due to imperfect homogeneity. In the event of molecular heterogeneity,

the dominant populations exhibit a masking effect on minor populations.

Single-molecule experiments

Single-molecule experiments, also known as in singulo methods, are designed to investigate the

characteristic properties one individual molecule at a time. A large number of individuals are studied

one at a time to satisfy statistical analysis for validity. Single-molecule experiments are effective to

detect subtle time-dependent conformational changes during catalysis and structural reorganization.

25 | P a g e

They yield a great deal of new information. Currently, several techniques including single-molecule

fluorescence and optical tweezers enabled biochemists to look into the workings of individual

molecules. The most notable example is mass spectrometry, where single ions are detected. Intrinsic

protein dynamics can also be studied within the confinement of biological nanopores. However, single

molecule experiments are limited by labeling and tethering requirements which might affect protein

dynamics.

Suppose an enzyme displays molecular heterogeneity, with three active forms that catalyze the same

reaction but at different rates. These forms have slightly different stabilities, but thermal noise is

sufficient to interconvert the forms. Each form is present as some fraction of the total enzyme

population. The value of KM under a particular set of conditions determined by many-molecules

experiments is the average value of all enzyme molecules present in the heterogeneous assembly.

However, a sufficient number of single-molecule experiments would reveal that the enzyme has three

different molecular forms with very different activities. These different forms would most likely

correspond to important biochemical differences.

The two most characterized enzymes are lysozyme and serine proteases.

Lysozyme

Lysozyme is an enzyme that destroys bacterial cell walls by hydrolyzing the β (1→4) glycosidic linkages

from N-acetylmuramic acid (NAM) to N-acetylglucosamine (NAG) in the alternating NAM–NAG

polysaccharide component of peptidoglycan cell wall. The X-ray structure of hen egg white (HEW)

lysozyme is the second structure of a protein and the first of an enzyme to be determined. Glu 35 acts as

an acid catalyst, and Asp 52 acts as a covalent catalyst. Hence Glu 35 and Asp 52 are lysozyme’s catalytic

residues.

Serine proteases

Proteolysis is the hydrolysis of a peptide (amide) bonds. Proteases catalyze proteolysis but with different

specificities. Papain, which is found in papaya, is quite undiscriminating; it will cleave any peptide bond

with little regard to the identity of the adjacent side chains. Serine proteases to promote a reaction that

is almost immeasurably slow at neutral pH in the absence of a catalyst. The best characterized serine

proteases are chymotrypsin, trypsin, and elastase. They are digestive enzymes that are synthesized by

the pancreatic acinar cells in inactive forms and secreted, via the pancreatic duct, into the duodenum

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(the small intestine’s upper loop). Serine proteases promote the reaction that is almost immeasurably

slow at neutral pH in the absence of proteolytic enzymes. Elastase is an enzyme that rapidly hydrolyzes

the nearly indigestible protein elastin. Elastin is a connective tissue protein rich with Ala, Gly, and Val

residues and rubberlike elastic properties. It is specific for small neutral residues.

Serine proteases have a common catalytic mechanism characterized by the presence of a peculiarly

reactive Ser residue that is essential for their enzymatic activity. The diagnostic test for the presence of

exceptionally active Ser residue is reaction with enzyme inactivating organophosphorus compounds

such as diisopropylphosphofluoridate (DIPF). Many organophosphorus compounds inactivate

acetylcholinesterase, a serine esterase that catalyzes the hydrolysis of acetylcholine. Hence, they are

potent nerve poisons. Acetylcholine transmits nerve impulses across the synapses (junctions) between

certain types of nerve cells. Therefore, acetylcholine is a neurotransmitter.

Zymogens

Many proteolytic enzymes are biosynthesized as larger inactive precursor forms known as zymogens or

proenzymes. If digestive enzymes were synthesized in their active forms, they would digest the tissues

that synthesized them. Zymogens are not enzymatically active as the catalytic residues are distorted.

Pancreatic trypsin inhibitor binds essentially irreversibly to any trypsin formed in the pancreas so as to

inactivate it. Besides, pancreatic zymogens are stored in intracellular vesicles called zymogen granules

whose membranous walls are believed to be resistant to enzymatic degradation.

The activation of trypsinogen, the zymogen of trypsin, occurs in two-step when trypsinogen enters the

duodenum from the pancreas. Enteropeptidase is a single-pass transmembrane serine protease located

in the duodenal mucosa. It specifically hydrolyzes trypsinogen’s Lys 15-Ile 16 peptide bond, thereby

excising its N-terminal hexapeptide. This yields the active enzyme, which has Ile 16 at its N-terminus.

The small amount of trypsin produced by enteropeptidase also catalyzes autocatalytic trypsinogen

activation, generating more trypsin. Thus, the formation of trypsin by enteropeptidase is the master

activation step since trypsin as the common activator of all the pancreatic zymogens.

Chymotrypsinogen is activated by the specific tryptic cleavage of its Arg 15-Ile 16 peptide bond to form

π-chymotrypsin. The π-chymotrypsin subsequently undergoes autolysis (self-digestion) to specifically

excise two dipeptides, Ser 14–Arg 15 and Thr 147–Asn 148, thereby yielding the equally active enzyme

α-chymotrypsin (or simply chymotrypsin). Chymotrypsin uses a histidine residue as a base catalyst to

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enhance the nucleophilic power of serine. Proelastase, the zymogen of elastase, is also activated by a

single tryptic cleavage to excise a short N-terminal polypeptide.

Acid proteases

Aspartic protease family (also known as acid proteases), are enzymes that contain catalytically essential

Asp residues. They include pepsin and chymosin. Pepsin is a digestive enzyme secreted by the stomach

and functions at pH 1. Pepsin was the first enzyme to be recognized (named in 1836 by Theodor

Schwann). Chymosin (formerly rennin) is a stomach enzyme which occurrs mainly in infants. It

specifically cleaves a Phe–Met peptide bond in the milk protein -casein, thereby causing milk to curdle,

making it easier to digest.

Drug design

Most drugs act by modifying the function of a particular protein in the body or in an invading pathogen.

The protein t may function as an enzyme, a transmembrane channel, or a receptor. Pharmacodynamics

is the biochemical and physiological effects of a drug and its mechanism of action. Drugs are discovered

by screening large numbers of synthetic compounds and natural products for the desired biochemical

effect. The first step is in vitro screening such as the degree of binding of a drug candidate to its target of

interest. This is followed by toxicity study toward the target bacteria or effects on a line of cultured

mammalian cells.

Cytochromes P450 functions in large part to detoxify xenobiotics and participate in the metabolic

clearance of the majority of drugs in use. It is a hemecontaining monooxygenase enzyme that occurs in

nearly all living organisms. In animals, P450 is embedded in the endoplasmic reticulum membrane.

Drug–drug interactions are often mediated by cytochrome P450. Toxicity and adverse reactions

eliminate most drug candidates. Common measures of the effect of a drug are the IC50, ED50, TD50, and

LD50. IC50 is the inhibitor concentration at which an enzyme exhibits 50% of its maximal activity. ED50 is

the effective dose of a drug required to produce a therapeutic effect in 50% of a test sample. TD50 is the

mean toxic dose required to produce a particular toxic effect in animals. LD50 is the mean lethal dose

required to kill 50% of a test sample. A drug candidate that exhibits a desired effect in the first two steps

is called a lead compound. A good lead compound binds to its target receptor with a dissociation

constant KD much lower than 1µM. Such a high affinity is necessary to minimize a drug’s less specific

binding to other macromolecules in the body and to ensure that only low doses of the drug need be

taken.

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Lead compound can be used as a point of departure to design more efficacious compounds by

systematic tools for drug discovery such as SAR and QSAR. Minor modifications to a drug candidate can

result in major changes in its pharmacological properties. Structure–activity relationships (SARs) is the

determination, via synthesis and screening, of which groups on a lead compound are important for its

drug function and which are not. Quantitative structure–activity relationship (QSAR) is based on the

premise that there is a relatively simple mathematical relationship between the biological activity of a

drug and its physicochemical properties. A QSAR can simultaneously take into account several

physicochemical properties of substituents such as their pK values, van der Waals radii, hydrogen

bonding energy, and conformation. The values of these parameters for each of the terms in a QSAR are

indicative of the contribution of that term to the drug’s activity. The use of QSARs to optimize the

biological activity of a lead compound is a valuable tool in drug discovery.

Structure-based drug design

Structure-based drug design (also called rational drug design) uses the structure of a receptor in

complex with a drug candidate to guide the development of more efficacious compounds. Structure-

based drug designing uses molecular modeling tools, quantum mechanical calculations and molecular

docking simulations. Molecular modeling computes the minimum energy conformation. Quantum

mechanical calculations determine charge distribution and potential electrostatic interaction of the

receptor with the ligand. Docking simulations computationally models the candidate inhibitor into the

binding site on the receptor.

Combinatorial chemistry and Fragment-based lead discovery

Combinatorial chemistry is a technique to rapidly and inexpensively synthesize large numbers of related

compounds. Combinatorial chemistry is combined with the development of robotics for high-throughput

screening of large numbers of drug libraries for substances that bind with high affinity to a drug target

(potential lead compounds). Combinatorial chemistry discovers a lead compound all at once. Fragment-

based lead discovery (FBLD) is a technique of screening only a relatively small number of simple

compounds for their ability to bind to the drug target with low affinity. Compounds that bind to a small

portion of the drug target’s surface area are then grown by adding chemical groups and/or linking

several such fragments together. FBLD discovers a lead compound one piece at a time.

Anti-AIDS agents

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Reverse transcriptase inhibitors are only partially effective. On the other hand, the development of

inhibitors of HIV-1 protease is one of the major successes of modern drug discovery methods. HIV-1

encodes two polyproteins, gag (55 kD) and gag–pol (160 kD), which are both anchored to the plasma

membrane. These polyproteins are cleaved by HIV-1 protease which is a member of the Asp proteases

family. Some HIV-1 protease inhibitors that are in clinical use include ritonavir, nelfinavir and indinavir.

HIV-1 protease inhibitors are very effective anti-AIDS agents.