INTRODUCTION – MECHANISM OF ENZYME CATALYSIS
- The enzymatic catalysis of reactions is essential to living systems. Under biologically relevant conditions, uncatalyzed reactions tend to be slow—most biological molecules are quite stable in the neutral-pH, mild-temperature, aqueous environment inside cells. Furthermore, many common chemical processes are unfavorable or unlikely in the cellular environment, such as the transient formation of unstable charged intermediates or the collision of two or more molecules in the precise orientation required for reaction.
- Reactions required to digest food, send nerve signals, or contract a muscle simply do not occur at a useful rate without catalysis. An enzyme avoid these problems by providing a specific environment in which a given reaction can occur more rapidly. The distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the confines of a pocket on the enzyme called the active site. The molecule that is bound in the active site and acted upon by the enzyme is called the substrate.
- The surface of the active site is lined with amino acid residues with substituent groups that bind the substrate and catalyze its chemical transformation. Often, the active site encloses a substrate, sequestering it completely from solution. The enzyme-substrate complex, an entity first proposed by Charles-Adolphe Wurtz in 1880, is central to the action of enzymes. It is also the starting point for mathematical treatments that define the kinetic behavior of enzyme-catalyzed reactions and for theoretical descriptions of enzyme mechanisms.
ENZYME SUBSTRATE BINDING MODEL
LOCK AND KEY MODEL
- It assumes that there is high degree of complementarity between enzyme and substrate (shape of substrate and the geometry of binding site on enzyme). The complementarity between enzyme and substrate is the basis of the lack and key model which was proposed by Emil fischer in 1894.
- It proposed that just the way any particular key can be fit to its particular lock in similar way substrate can only bind to its respective enzyme.
INDUCED FIT THEORY
According to this theory, enzymes are flexible and that the shape of active site can be markedly modified by binding of substrate. The binding of substrate induces conformational change in the enzyme that result in the complementary fit once the substrate bond. The binding site has different three dimensional shape before the substrate is bound.
RATE OF REACTION
A simple enzymatic reaction might be written
where E, S, and P represent the enzyme, substrate, and product; ES and EP are transient complexes of the enzyme with the substrate and with the product.
There is an energy barrier between S and P: the energy required for alignment of reacting groups, formation of transient unstable charges, bond rearrangements, and other transformations required for the reaction to proceed in either direction. This is called the transition state.
The transition state is not a chemical species with any significant stability and should not be confused with a reaction intermediate (such as ES or EP). It is simply a molecular moment in which events such as bond breakage, bond formation, and charge development have proceeded to the precise point at which decay to substrate or decay to product are equally likely. The difference between the energy levels of the ground state and the transition state is the activation energy, ΔG‡ .
The rate of a reaction reflects this activation energy: a higher activation energy corresponds to a slower reaction. Reaction rates can be increased by raising the temperature and/or pressure, thereby increasing the number of molecules with sufficient energy to overcome the energy barrier. Alternatively, the activation energy can be lowered by adding a catalyst. Catalysts enhance reaction rates by lowering activation energies.
Enzymes are no exception to the rule that catalysts do not affect reaction equilibria. The role of enzymes is to accelerate the interconversion of S and P. The enzyme is not used up in the process, and the equilibrium point is unaffected. However, the reaction reaches equilibrium much faster when the appropriate enzyme is present, because the rate of the reaction is increased.
This general principle is illustrated in the conversion of sucrose and oxygen to carbon dioxide and water:
This conversion which takes place through a series of separate reactions, has a very large and negative ΔG′°, and at equilibrium the amount of sucrose present is negligible. Yet sucrose is a stable compound, because the activation energy barrier that must be overcome before sucrose reacts with oxygen is quite high. Sucrose can be stored in a container with oxygen almost indefinitely without reacting. In cells, however, sucrose is readily broken down to CO2 and H2O in a series of reactions catalyzed by enzymes.
These enzymes not only accelerate the reactions, they organize and control them so that much of the energy released is recovered in other chemical forms and made available to the cell for other tasks. The reaction pathway by which sucrose (and other sugars) is broken down is the primary energy-yielding pathway for cells, and the enzymes of this pathway allow the reaction sequence to proceed on a biologically useful time scale.
Any reaction may have several steps, involving the formation and decay of transient chemical species called reaction intermediates. A reaction intermediate is any species on the reaction pathway that has a finite chemical lifetime (longer than a molecular vibration, ~10 −13 second).
When the S ⇌ P reaction is catalyzed by an enzyme, the ES and EP complexes can be considered intermediates. Additional, less-stable chemical intermediates often exist in the course of an enzyme-catalyzed reaction. The interconversion of two sequential reaction intermediates thus constitutes a reaction step. When several steps occur in a reaction, the overall rate is determined by the step (or steps) with the highest activation energy; this is called the rate-limiting step. In a simple case, the rate-limiting step is the highest-energy point in the diagram for interconversion of S and P.
In practice, the rate-limiting step can vary with reaction conditions, and for many enzymes several steps may have similar activation energies, which means they are all partially rate-limiting. Activation energies are energy barriers to chemical reactions.
These barriers are crucial to life itself. The rate at which a molecule undergoes a particular reaction decreases as the activation barrier for that reaction increases. Without such energy barriers, complex macromolecules would revert spontaneously to much simpler molecular forms, and the complex and highly ordered structures and metabolic processes of cells could not exist. Over the course of evolution, enzymes have developed to lower activation energies selectively for reactions that are needed for cell survival.
Enzyme commonly employs one or more strategies to catalyze specific reaction. Among the best characterized mechanism are general acid base catalysis, metal ion catalysis and covalent catalysis. A combination of several catalytic strategies is usually employed by an enzyme.
The process of covalent catalysis involves the formation of a reversible covalent bond between enzyme and substrate. Although a covalent interaction are temporary the substrate is bound to enzyme during the course of catalysis. After the completion of reaction the enzyme returns to original unmodified state. The proteolytic enzyme chemotrypsin provides an excellent example of this mechanism.
Enzyme performing covalent catalysis bear nucleophillic or electrophillic centers. Nucleophillic centers includes amine, carboxyl, hydroxyl, imidazole and thiol group. These group perform covalent nucleophillic catalysis and readily effects electrophillic centers of substrate forming covalent intermediates. Typical electrophillic centers in substrates includes phosphoryl group, acyl group and glycosyl group.
Covalent electrophillic catalysis is also observed but usually involves coenzyme adduct that generates elecrophillic centers.
Acid base catalysis
Acid base catalysis involves proton transfer. There are two kinds of acid base catalysis specific and general. In specific acid base catalysis, hydronium and hydroxide ions acts directly as acid and base group. In general acid base catalysis donor and acceptor of proton are other than hydronium and hydroxide ion. In such cases the ionizable functional group of aminoacyl side chain and of cofactor (if present) contribute to catalysis by acting as acid or base.
Functional group such as imidazole, hydroxyl, carboxyl, sulfhydral, amino and phenolic group of amino acid acts as acid or base as far as enzyme catalyst reaction are concerned, only general acid base catalyst can occur. A common example of general acid base reaction occur in hydrolysis of peptidoglycan catalysis by lysozyme. Specific acid and base catalysis is determined by pH and buffer, concentration has no effect.
Metal ion catalysis ( lewis acid base catalysis)
Many enzymes requires metal ion for their catalytic activity. These enzymes can be metalloenzymes ( enzyme bind to metal ion very tightly or require the metal ion to maintain its stable native state) or metal activated ( bind metal ion weakly during catalysis).
Metal ion can function catalytically in several ways. One role of metal ion in metal activated enzymes and metalloenzyme is to act as electrophillic catalyst, stabilizing the increase electron density of negative charge on reaction intermediate that can develop during reaction. Metal can also mediate oxidation reduction reaction by reversible changes in the metal ion oxidation state.
Amount of enzymes can either be expressed as molar amounts or measure in terms of activity. Enzymes are usually present in very small quantity. When enzyme are quantified relative to their activity rather than direct measurement of their concentration the unit used to report enzyme level are activity units. There are two standard unit to express enzyme activity
Enzyme unit the amount of enzyme causing transformation of 1 micro mole of substance per minute at 25 degree C under optimal condition of measurement
1 enzyme unit = I micro mole per minute
Katal the catal is accepted S.I unit of enzyme activity. 1 katal is amount of enzyme that catalyzes the transformation of one mole of substrate per second.
1 katal = 1 mole per second.
- Enzymes are highly effective catalysts, commonly enhancing reaction rates by a factor of 105 to 1017 .
- Enzyme-catalyzed reactions are characterized by the formation of a complex between substrate and enzyme (an ES complex). Substrate binding occurs in a pocket on the enzyme called the active site.
- The function of enzymes and other catalysts is to lower the activation energy, ΔG‡ , for a reaction and thereby enhance the reaction rate. The equilibrium of a reaction is unaffected by the enzyme.
- A significant part of the energy used for enzymatic rate enhancements is derived from weak interactions (hydrogen bonds, aggregation due to the hydrophobic effect, and ionic interactions) between substrate and enzyme. The enzyme active site is structured so that some of these weak interactions occur preferentially in the reaction transition state, thus stabilizing the transition state.
- The need for multiple interactions is one reason for the large size of enzymes. The binding energy, ΔGB, is used to offset the energy required for activation, ΔG‡ , in several ways—for example, lowering substrate entropy, causing substrate desolvation, or causing a conformational change in the enzyme (induced fit). Binding energy also accounts for the exquisite specificity of enzymes for their substrates.
- Additional catalytic mechanisms employed by enzymes include general acid-base catalysis, covalent catalysis, and metal ion catalysis. Catalysis often involves transient covalent interactions between the substrate and the enzyme, or group transfers to and from the enzyme, to provide a new, lower- energy reaction path. In all cases, the enzyme reverts to the unbound state once the reaction is complete
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