ENZYMES AND CATALYSISENZYMES AND CATALYSIS

Dr. Muhammad Zeeshan HyderDr. Muhammad Zeeshan Hyder

Dept of Biosciences, CIIT IslamabadDept of Biosciences, CIIT Islamabad

Chapter 8BIOCHEMISTRY, by Lubert Stryer 5th Edition 11

Enzymes are the catalysts of biological systems They also mediate the transformation of one form of

energy into another. The most striking characteristics of enzymes are their

catalytic power and specificity. Catalysis takes place at a particular site on the enzyme called the active site.

Nearly all known enzymes are proteins. However, the discovery of catalytically active RNA molecules provides compelling evidence that RNA was an early biocatalyst.

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By utilizing the full repertoire of intermolecular forces, enzymes bring substrates together in an optimal orientation, the prelude to making and breaking chemical bonds.

They catalyze reactions by stabilizing transition states, the highest-energy species in reaction pathways.

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Enzymes Are Powerful and Highly Specific Catalysts

Enzymes accelerate reactions by factors of as much as a million or more (Table 8.1). Indeed, most reactions in biological systems do not take place at perceptible rates in the absence of enzymes. Even a reaction as simple as the hydration of carbon dioxide is catalyzed by an enzyme namely, carbonic anhydrase.

The transfer of CO2 from the tissues into the blood and then to the alveolar air would be less complete in the absence of this enzyme. In fact, carbonic anhydrase is one of the fastest enzymes known. Each enzyme molecule can hydrate 106 molecules of CO2 per second. This catalyzed reaction is 107 times as fast as the uncatalyzed one.

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Enzymes are highly specific both in the reactions that they catalyze and in their choice of reactants, which are called substrates.

An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions. Side reactions leading to the wasteful formation of by-products are rare in enzyme-catalyzed reactions, in contrast with uncatalyzed ones.

The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme. This precision is a result of the intricate three-dimensional structure of the enzyme protein.

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Many Enzymes Require Cofactors for Activity

The catalytic activity of many enzymes depends on the presence of small molecules termed cofactors, although the precise role varies with the cofactor and the enzyme. Such an enzyme without its cofactor is referred to as an apoenzyme; the complete, catalytically active enzyme is called a holoenzyme.

Cofactors can be subdivided into two groups: metals and small organic molecules. The enzyme carbonic anhydrase, for example, requires Zn2+ for its activity.

Glycogen phosphorylase which mobilizes glycogen for energy, requires the small organic molecule pyridoxal phosphate (PLP).Cofactors that are small organic molecules are called coenzymes. Often derived from vitamins, coenzymes can be either tightly or loosely bound to the enzyme. If tightly bound, they are called prosthetic groups.

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Many Enzymes Require Cofactors for Activity

Loosely associated coenzymes are more like cosubstrates because they bind to and are released from the enzyme just as substrates and products are. The use of the same coenzyme by a variety of enzymes and their source in vitamins sets coenzymes apart from normal substrates, however. Enzymes that use the same coenzyme are usually mechanistically similar.

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In many biochemical reactions, the energy of the reactants is converted with high efficiency into a different form.

For example, in photosynthesis, light energy is converted into chemical-bond energy through an ion gradient.

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.

Enzymes may then use the chemical-bond energy of ATP in many ways. The enzyme myosin converts the energy of ATP into the mechanical energy of contracting muscles. Pumps in the membranes of cells and organelles, which can be thought of as enzymes that move substrates rather than chemically altering them, create chemical and electrical gradients by using the energy of ATP to transport molecules and ions.

Enzymes May Transform Energy from One Form into Another

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Enzymes Are Classified on the Basis of the Types of Reactions That They

Catalyze Many enzymes have common names that provide little

information about the reactions that they catalyze. For example, a proteolytic enzyme secreted by the

pancreas is called trypsin. Most other enzymes are named for their substrates and for the reactions that they catalyze, with the suffix "ase" added. Thus, an ATPase is an enzyme that breaks down ATP,whereas ATP synthase is an enzyme that synthesizes ATP.

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To bring some consistency to the classification of enzymes, in 1964 the International Union of Biochemistry established an Enzyme Commission to develop a nomenclature for enzymes. Reactions were divided into six major groups.

These groups were subdivided and further subdivided, so that a four-digit number preceded by the letters EC for Enzyme Commission could precisely identify all enzymes.

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Consider as an example nucleoside monophosphate (NMP) kinase. It catalyzes the following reaction:

NMP kinase transfers a phosphoryl group from ATP to NMP to form a nucleoside diphosphate (NDP) and ADP. Consequently, it is a transferase, or member of group 2.

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Many groups in addition to phosphoryl groups, such as sugars and carbon units, can be transferred. Transferases that shift a phosphoryl group are designated 2.7.

Various functional groups can accept the phosphoryl group. If a phosphate is the acceptor, the transferase is designated 2.7.4.

The final number designates the acceptor more precisely. In regard to NMP kinase, a nucleoside monophosphate is the acceptor, and the enzyme's designation is EC 2.7.4.4.

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Enzymes Alter Only the Reaction Rate and Not the Reaction Equilibrium

An enzyme cannot alter the laws of thermodynamics and consequently cannot alter the equilibrium of a chemical reaction. This inability means that an enzyme accelerates the forward and reverse reactions by precisely the same factor.

Consider the interconversion of A and B. Suppose that, in the absence of enzyme, the forward rate constant (k F) is 10-4 s-1 and the reverse rate constant (k R) is 10-6 s-1.

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The equilibrium concentration of B is 100 times that of A, whether or not enzyme is present. However, it might takeconsiderable time to approach this equilibrium without enzyme, whereas equilibrium would be attained rapidly in the presence of a suitable enzyme.

Enzymes accelerate the attainment of equilibria but do not shift their positions. The equilibrium position is a function only of the free-energy difference between

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Enzymes Accelerate Reactions by Facilitating the Formation of the Transition

State A chemical reaction of substrate S to form product P goes through a

transition state S that has a higher free energy than does either S or P. The double dagger denotes a thermodynamic property of the transition state.

The transition state is the most seldom occupied species along the reaction pathway because it is the one with the highest free energy.

The difference in free energy between the transition state and the substrate is called the Gibbs free energy of activation or simply the activation energy, symbolized by D G

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The energy of activation, or ΔG , does not enter into the final ΔG calculation for the reaction, because the energy input required to reach the transition state is returned when the transition state forms the product.

The activation energy barrier immediately suggests how enzymes enhance reaction rate without altering ΔG of the reaction: enzymes function to lower the activation energy, or, in other words, enzymes facilitate the formation of the transition state.

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The Formation of an Enzyme-Substrate Complex Is the First Step in Enzymatic

Catalysis

The substrates are bound to a specific region of the enzyme called the active site. Most enzymes are highly selective in the substrates that they bind.

Indeed, thecatalytic specificity of enzymes depends in part on the specificity of binding.

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The Active Sites of Enzymes Have Some Common Features

The active site of an enzyme is the region that binds the substrates (and the cofactor, if any). It also contains the residues that directly participate in the making and breaking of bonds. These residues are called the catalytic groups.

In essence, the interaction of the enzyme and substrate at the active site promotes the formation of the transition state. The active site is the region of the enzyme that most directly lowers the D G of the reaction, which results in the rate enhancement characteristic of enzyme action.

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1. The active site is a three-dimensional cleft formed by groups that come from different parts of the amino acid sequence

2. The active site takes up a relatively small part of the total volume of an enzyme.

3. Active sites are clefts or crevices. 4. Substrates are bound to enzymes by multiple

weak attractions. 5. The specificity of binding depends on the

precisely defined arrangement of atoms in an active site.

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Binding of enzyme with its Binding of enzyme with its substratesubstrate

The binding of enzyme with its substrate was proposed The binding of enzyme with its substrate was proposed initially as “lock and key” initially as “lock and key”

Now we know that enzymes are flexible and their shape is Now we know that enzymes are flexible and their shape is markedly changed when the bind to their substrate.markedly changed when the bind to their substrate.

This mode of binding is called as “induced fit”This mode of binding is called as “induced fit”

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Most Biochemical Reactions Include Multiple Substrates

Sequential Displacement. In the sequential mechanism, all substrates must bind to the enzyme

before any product is released. Consequently, in a bisubstrate reaction, a ternary complex of the enzyme and both substrates forms.

Sequential mechanisms are of two types: ordered, in which the substrates bind the enzyme in a defined sequence, and random.

Many enzymes that have NAD+ or NADH as a substrate exhibit the sequential ordered mechanism.

Consider lactate dehydrogenase, an important enzyme in glucose metabolism. This enzyme reduces pyruvate to lactate

while oxidizing NADH to NAD+.

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In the ordered sequential mechanism, the coenzyme always binds first and the lactate is always released first. This sequence can be represented as follows in a notation developed by W. Wallace Cleland:

The enzyme exists as a ternary complex: first, consisting of the enzyme and substrates and, after catalysis, the enzyme and products.

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In the random sequential mechanism, the order of addition of substrates and release of products is random.

Sequential random reactions are illustrated by the formation of phosphocreatine and ADP from ATP and creatine, a reaction catalyzed by creatine kinase

Although the order of certain events is random, the reaction still passes through the ternary complexes including, first, substrates and, then, products.

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Double-Displacement (Ping-Pong) Reactions.

In double-displacement, or Ping-Pong, reactions, one or more products are released before all substrates bind the enzyme. The defining feature of double-displacement reactions is the existence of a substituted enzyme intermediate, in which the enzyme is temporarily modified. Reactions that shuttle amino groups between amino acids and a-keto acids are classic examples of double-displacement mechanisms.

The enzyme aspartate aminotransferase catalyzes the transfer of an amino group from aspartate to a-ketoglutarate.

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After aspartate binds to the enzyme, the enzyme removes aspartate's amino group to form the substituted enzyme

intermediate. The first product, oxaloacetate, subsequently departs. The second substrate, a-ketoglutarate, binds to the

enzyme, accepts the amino group from the modified enzyme, and is then released as the final product, glutamate.

In the Cleland notation, the substrates appear to bounce on and off the enzyme analogously to a Ping-Pong ball bouncing on a table.

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Enzymes Can Be Inhibited by Specific Molecules

The activity of many enzymes can be inhibited by the binding of specific small molecules and ions.

This means of inhibiting enzyme activity serves as a major control mechanism in biological systems.

The regulation of allosteric enzymes typifies this type of control.

In addition, many drugs and toxic agents act by inhibiting enzymes.

Inhibition by particular chemicals can be a source of insight into the mechanism of enzyme action: specific inhibitors can often be used to identify residues critical for catalysis.

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Enzyme inhibition can be either reversible or irreversible.

An irreversible inhibitor dissociates very slowly from its target enzyme because it has become tightly bound to the enzyme, either covalently or noncovalently.

Some irreversible inhibitors are important drugs.

Penicillin acts by covalently modifying the enzyme transpeptidase, thereby preventing the synthesis of bacterial cell walls and thus killing the bacteria.

Aspirin acts by covalently modifying the enzyme cyclooxygenase, reducing the synthesis of inflammatory signals.

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Reversible inhibition, in contrast with irreversible inhibition, is characterized by a rapid dissociation of the enzyme-inhibitor complex.

In competitive inhibition, an enzyme can bind substrate (forming an ES complex) or inhibitor (EI) but not both (ESI).

The competitive inhibitor resembles the substrate and binds to the active site of the enzyme.

The substrate is thereby prevented from binding to the same active site.

A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate. 3030

In noncompetitive inhibition, which also is reversible, the inhibitor and substrate can bind simultaneously to an enzyme molecule at different binding sites.

A noncompetitive inhibitor acts by decreasing the turnover number rather than by diminishing the proportion of enzyme molecules that are bound to substrate.

Noncompetitive inhibition, in contrast with competitive inhibition, cannot be overcome by increasing the substrate concentration.

A more complex pattern, called mixed inhibition, is produced when a single inhibitor both hinders the binding of substrate and decreases the turnover number of the enzyme.

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Vitamins Are Often Precursors to Coenzymes

Vitamins are small biomolecules that are needed in small amounts in the diets of higher animals.

The water-soluble vitamins are vitamin C (ascorbate, an antioxidant) and the vitamin B complex (components of coenzymes).

Ascorbate is required for the hydroxylation of proline residues in collagen, a key protein of connective tissue.

The fat-soluble vitamins are vitamin A (a precursor of retinal), D (a regulator of calcium and phosphorus metabolism), E (an antioxidant in membranes), and K (a participant in the carboxylation of glutamate).

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A Few Basic Catalytic Principles Are Used by Many Enzymes

Enzymes commonly employ one or more of the following strategies to catalyze specific reactions:

1. Covalent catalysis. In covalent catalysis, the active site contains a reactive group, usually a powerful nucleophile that becomes temporarily covalently modified in the course of catalysis.

The proteolytic enzyme chymotrypsin provides an excellent example of this mechanism.

2. General acid-base catalysis. In general acid-base catalysis, a molecule other than water plays the role of a proton donor or acceptor.

Chymotrypsin uses a histidine residue as a base catalyst to enhance the nucleophilic power of serine

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3. Metal ion catalysis. Metal ions can function catalytically in several ways. For instance, a metal ion may serve as an electrophilic catalyst, stabilizing

a negative charge on a reaction intermediate. Alternatively, the metal ion may generate a nucleophile by increasing the

acidity of a nearby molecule, such as water in the hydration of CO2 by carbonic anhydrase.

Finally, the metal ion may bind to substrate, increasing the number of interactions with the enzyme and thus the binding energy.

This strategy is used by NMP kinases.

4. Catalysis by approximation. Many reactions include two distinct substrates.

In such cases, the reaction rate may be considerably enhanced by bringing the two substrates together along a single binding surface on an enzyme.

NMP kinases bring two nucleotides together to facilitate the transfer of a phosphoryl group from one nucleotide to the other.

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Thank you

Two views of the adenovirus protease, an enzyme required for viral replication.

DNA

protease

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