Module II

Enzyme and Enzyme Kinetics

Dr V Gaanapriya
Enzymes- Introduction
• A cell must be capable of performing a multitude of chemical changes in order to stay alive, grow and
reproduce.
• It may have to alter complex nutrients in the medium before they can enter the cell.
• These nutrients are broken down chemically to provide energy for the cell and precursors for the
synthesis of cell material.
• Enzymes present in the cell are minute and capable of speeding up the chemical reactions associated
with the life processes.
• Any impairment of enzymes and their activity are reflected by some changes in the cell, or even by
death.
• Hence, there can be no life without enzymes.
Enzyme
• Enzymes are usually proteins of high molecular weight (15,000  MW  several million daltons) that
act as catalysts
• Recently, it has been shown that some RNA molecules are also catalytic, but the vast majority of
cellular reactions are mediated by protein catalysts. RNA molecules that have catalytic properties
are called ribozymes.
• Enzymes are specific, versatile, and very effective biological catalysts, resulting in much higher
reaction rates as compared to chemically catalyzed reactions under ambient conditions.
• More than 2000 enzymes are known. Enzymes are named by adding the suffix -ase to the end of the
substrate, such as urease, or the reaction catalyzed, such as alcohol dehydrogenase.
• Some enzymes have a simple structure, such as a folded polypeptide chain (typical of most hydrolytic
enzymes). Many enzymes have more than one subunit.
• Some protein enzymes require a nonprotein group for their activity. This group is either a cofactor,
such as metal ions, Mg, Zn, Mn, Fe, or a coenzyme, such as a complex organic molecule, NAD, FAD,
CoA, or some vitamins.
Enzyme
• An enzyme containing a non-protein group is called a holoenzyme (functional unit of enzyme).
• The protein part of this enzyme is the apoenzyme (holoenzyme = apoenzyme + cofactor).
• Enzymes that occur in several different molecular forms, but catalyze the same reaction, are called
isozymes.
• Some enzymes are grouped together to form enzyme complexes.
• Enzymes are substrate specific and are classified according to the reaction they catalyze.
• Enzymes function in sequences of reaction called pathways.
• For a cell to grow, normally it is essential that the flow of chemical substances or the metabolites
through these pathways be under a high degree of regulation or control.
• This regulation ensures that no products are deficient or in excess exerted either on enzyme activity or
on the enzyme synthesis.
How do enzymes work
Enzyme
• Enzymes are biological catalysts synthesized by the living cells.
• They are protein in nature, colloidal and specific in their action.
• Enzymes are thermo labile in character, for example the hydrolysis of proteins by a strong acid at
100°C may require a couple of days, but by using enzymes, the proteins are fully digested in the
gastrointestinal tract at body temperature in just a few hours.
• Also in a bacterial cell, hundreds of chemical reactions occur which lead to cell division in a few
minutes, whereas the reactions taking place outside the cell will take a couple of days.
• The very existence of life is unimaginable without the presence of enzymes.
Classification of Enzymes
In the year 1961, the Enzyme Commission, IUB (International Union of Biochemists) proposed six
major classes. Each class on its own represents the general type of reaction.
[Link]: They function in the oxidation-reduction reactions.
[Link]: They catalyze the transfer of the functional groups.
[Link]: They bring about the hydrolysis of compounds.
[Link]: They are specialized in the addition or removal of H2O, NH3, CO2, etc.
[Link]: They are used in the isomerization reactions.
[Link]: They catalyze the synthetic reactions where two molecules are joined together.
The other way of classifying is: intracellular enzymes and extracellular enzymes.
The intracellular enzymes are functional within the cells where they are synthesized, whereas the
extracellular enzymes are active outside the cells, for example, digestive enzymes like trypsin, pepsin, etc.
Classification of Enzymes
 Enzyme catalysis
• A catalyst is a substance which increases the rate of a chemical reaction without itself undergoing a permanent chemical change.
• It only influences the rate of a chemical reaction; it does not affect the reaction equilibrium.
• Enzymes lower the activation energy of the reaction catalyzed by binding the substrate and forming an enzyme–substrate complex.
Enzymes do not affect the free-energy change or the equilibrium constant.
• Overall it is seen that catalytic reactions are much faster than the non- catalytic reactions. K = A exp(-Ea/RT)
Enzyme catalysis
• The interaction varies from one enzyme–substrate complex to another.
• Various studies using X-ray and Raman spectroscopy have revealed the presence of the enzyme–
substrate (ES) complex. The interaction between the enzyme and its substrate is usually by weak
forces.
• In most cases, van der Waals forces and hydrogen bonding are responsible for the formation of ES
complexes. The substrate binds to a specific site on the enzyme known as the active site.
• The substrate is a relatively small molecule and fits into a certain region on the enzyme molecule,
which is a much larger molecule.
• The simplest model describing this interaction is the lock-and-key model, in which the enzyme
represents the lock and the substrate represents the key
Enzyme catalysis
• In multisubstrate enzyme-catalyzed reactions, enzymes can hold substrates such that reactive
regions of substrates are close to each other and to the enzyme’s active site, which is known as the
proximity effect.
• Also, enzymes may hold the substrates at certain positions and angles to improve the reaction rate,
which is known as the orientation effect.
• In some enzymes, the formation of an enzyme–substrate complex causes slight changes in the three-
dimensional shape of the enzyme.
• This induced fit of the substrate to the enzyme molecule may contribute to the catalytic activity of
the enzyme, too. The enzymes lysozyme and carboxypeptidase A have been observed to change their
three-dimensional structure upon complexing with the substrate.
• Enzyme catalysis is affected not only by the primary structure of enzymes but also by the secondary,
tertiary, and quaternary structures.
• The properties of the active site of enzymes and the folding characteristics have a profound effect on
the catalytic activity of enzymes. Certain enzymes require coenzymes and cofactors for proper
functioning.
Order of a reaction
• Consider a non-enzymatic reaction, S→P ,where S = substrate and P = product.
• The rate or velocity of a reaction (v), varies directly with concentration of the reactants, whether substrate or
catalyst. The rate is given as follows:
Δ𝑃
• Δ𝑡
= v = 𝑘[S]
• where k is the rate constant that indicates the speed or the efficiency of a reaction

• The overall kinetic order of a reaction is the sum of the exponents in the equation, i.e. as to how many molecules are
reacting in the rate-determining step. Therefore, S→P is a first order reaction.
• Now, consider
• S1 + S2 → P1 + P2
• It is a single step reaction. In this case, the rate is found by knowing the concentrations of both the reactants, i.e. the
substrates. If S1 and S2 are present in similar concentrations, the rate equation is
1[ 1
• v = k[S1] S2]
 Order of a reaction
• With respect to each reactant, the reaction is the first order and overall it is a second order or a
bimolecular reaction.
• If [S2] >> [S1], so that it remains essentially constant, the reaction is a zero order reaction with respect
to that reactant, i.e. [S2]. So [S2] is unchanging and can be eliminated. Therefore,
1 0
• v = k[S1] [S2] or
• v = k’[S1]
• For example, in the non-enzymatic hydrolysis of sucrose in the aqueous acid solutions,
• C12H22O11 + H2O →C6H12O6 + C6H12O6

• Here [H2O] >>[S1] and [H2O] is constant. So the rate depends only on the concentration of sucrose, i.e.
[S1].
• Now let us consider purely enzymatic reactions and the various mechanisms of interaction of
substrates and formation of products.
Order of a reaction
• Generally, an enzymatic reaction is given as follows: E + S→ES →E + P
• where E = enzyme; S = substrate
ES = enzyme=substrate complex ;P = product
• The prime requisite for the enzyme catalysis is that S must combine with E at the active site to form
ES, the enzyme-substrate complex. Once combined, after sometime form the products with the
liberation of E.
• More specifically, it is represented as E + S ⇌ ES →E + P which shows that the formation of ES
complex is reversible and the formation of the products is irreversible.
• The idea of ES complex formation was first conceived by Michaelis-Menten in the year 1913 and has
been widely accepted.
• A few theories have been proposed to explain the formation of ES complex.
Order of a reaction
• Emil Fischer proposed that E is a rigid template and S is a matching key.
• The rate of enzymatic reactions depends on the concentration of both the substrate and enzyme.
• When the amount of S is high enough or when the amount of E is much less than the S, then the reaction is the pseudo
first order, i.e.
1 0 1
• V = k[E] [S] or V = k[E] or V = k’[E]
• It shows that the more is enzyme present, the faster is the reaction. Increase in the concentration of enzymes the rate
increases.
•Note: [S] is high and fixed, i.e. at saturation throughout the reaction.
•Pseudo first order reactions are used in the analysis called enzyme assays that determine the concentration of
enzymes.
Progress of a Reaction
At the beginning of a reaction E + S→ES →E + P, the amount of product P formed is very low and so it is negligible. More
specifically now the reaction is represented as

•where the rate constants

k1 = rate of association of E with S
• k-1 = rate of dissociation of S from E
• kcat = catalytic constant , also called Turn Over Number (TON).
• The TON is defined as the number of substrate molecules that can be converted by a molecule of an enzyme in a
minute. Also defined as the number of catalytic events per second per enzyme molecule. The TON varies from 100 to 3
× 106.
• The conversion of ES complex to E + P is shown by a single one way arrow.
• During the initial stages when the measurements are made, little product is formed so the rate of reverse reaction is
negligible. The velocity measured during this period, which is in fact very short, is called initial velocity, V0.
• The formation of ES complex and the dissociation of ES complex are in fact rapid whereas the conversion of S to product
is usually slow. Hence, it is the rate limiting step. In this step, the substrate is chemically altered.
Progress of a Reaction
The curves for [P] vs time (t) for different enzyme concentrations. The lines drawn to the curve indicate the initial
period, which is at the lower portion of the curve and given as
Δ𝑃
= V0
Δ𝑡
As the reaction proceeds, [P] increases. The rate of reaction doubles when twice as much enzyme (2E) is added to
another identical reaction mixture.
Enzyme Kinetics
• Kinetics of simple enzyme-catalyzed reactions are often referred to as Michaelis–Menten kinetics or
saturation kinetics.

• The qualitative features of enzyme kinetics are similar to Langmuir–Hinshelwood kinetics .

• These models are based on data from batch reactors with constant liquid volume in which the initial
substrate, [S0], and enzyme, [E0], concentrations are known.
• More complicated enzyme-substrate interactions such as multisubstrate–multienzyme reactions can
take place in biological systems.
• An enzyme solution has a fixed number of active sites to which substrates can bind.
• At high substrate concentrations, all these sites may be occupied by substrates or the enzyme is
saturated.
• Saturation kinetics can be obtained from a simple reaction scheme that involves a reversible step for
enzyme–substrate complex formation and a dissociation step of the ES complex.
Enzyme Kinetics
• It is assumed that the ES complex is established rather rapidly and the rate of the reverse reaction of
the second step is negligible.
• The assumption of an irreversible second reaction often holds only when product accumulation is
negligible at the beginning of the reaction.
• Two major approaches used in developing a rate expression for the enzyme- catalyzed reactions are (1)
rapid-equilibrium approach and (2) quasi-steady-state approach.