enzymes subtopics
TRANSCRIPT
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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
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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.
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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.