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Reaction path

Reaction path

FIGURE 7.34 Saturation Kinetics

As the substrate concentration increases, the velocity of the reaction reaches a maximum (Vm). The Km is the substrate concentration that yields half the maximum velocity.

The maximum velocity and the Michaelis constant summarize an enzymes kinetic properties.

however, the metal ion is not reduced or oxidized, but acts to stabilize negative charges on the reaction intermediate. Thus Zn2+ in the active site of car-boxypeptidase polarizes the C=O of the peptide bond which is about to be broken.

6. Distortion of the substrate—(up to 108 fold). Binding to the enzyme may distort the substrate. The active site of the enzyme may fit the transition state intermediate better than the substrate. Once the substrate is bound it will be forced into the shape of the reaction intermediate. Distortion is difficult to demonstrate since the strain is imposed only after the substrate has bound to the enzyme.

The Rate of Enzyme Reactions

The principles of chemical kinetics apply to enzyme reactions, with certain modifications. Typically, reaction rates are proportional to the concentrations of the reactants. For enzymes, the rate is proportional to substrate concentration, [S], only at low substrate concentrations. At higher substrate concentrations all the active sites of the enzyme will be filled by substrate and the enzyme can work no faster. The enzyme is said to be saturated and has reached its maximum velocity, or Vmax or Vm. The resulting relation between [S] and rate is a hyperbolic curve (Fig. 7.34).

The Vmax depends on the nature of the chemical reaction and hence depends on the chemical properties of the substrate and the enzyme active site. The Vmax may vary with pH and temperature. The more enzyme, the more substrate it can handle, so Vmax is also proportional to the amount of enzyme. The Km, or Michaelis constant, is the substrate concentration that gives half maximal velocity. It largely depends on the affinity of the substrate for the active site and is independent of the enzyme concentration. The lower the Km, the higher the affinity of the substrate for the enzyme and the faster the reaction (— at low substrate concentrations; at high substrate concentrations the Km becomes irrelevant).These factors are summarized in the MichaelisMenten equation:

Substrate analogs bind to the enzyme active site instead of the substrate. Some inhibit the enzyme, others react in a way similar to the natural substrate.

Substrate Analogs and Enzyme Inhibitors Act at the Active Site

Analogs are molecules resembling natural substances sufficiently well to bind to the active site of the enzyme. Some analogs act as alternative substrates for the enzyme. Other analogs bind to the active site, but instead of reacting, they block the active site and inhibit the enzyme. Such analogs are known as competitive inhibitors, as they compete with the true substrate for binding to the enzyme. The extent of inhibition depends both on the relative concentrations and the relative affinities of the substrate and the inhibitor.

The active site of many, perhaps most, enzymes is designed to better fit the reaction intermediate or transition state, rather than the substrate itself. This tends to analog A chemical substance that mimics another well enough to be mistaken for it by biological macromolecules, in particular enzymes, receptor proteins, or regulatory proteins competitive inhibitor Chemical substance that inhibits an enzyme by mimicking the true substrate well enough to be mistaken for it Km See Michaelis constant maximum velocity (Vm or Vmax) Velocity reached when all the active sites of an enzyme are filled with substrate

Michaelis constant (Km) The substrate concentration that gives half maximal velocity in an enzyme reaction. It is an inverse measure of the affinity of the substrate for the active site Michaelis-Menten equation Equation describing relationship between substrate concentration and the rate of an enzyme reaction saturated (Referring to enzymes) When all the active sites are filled with substrate and the enzyme cannot work any faster

Substrate Analogs and Enzyme Inhibitors Act at the Active Site 185

FIGURE 7.35 Proline Racemase Produces a Planar Intermediate

A) In the conversion of the L-isomer to the D-isomer of proline, the intermediate product is flat. This reaction is reversible. B) The best analogs for inhibiting the reaction are planar since these most closely resemble the flat transition state intermediate.

Substrate Analogs and Enzyme Inhibitors Act at the Active Site 185

B) Production of analogs as inhibitors

FIGURE 7.35 Proline Racemase Produces a Planar Intermediate

A) In the conversion of the L-isomer to the D-isomer of proline, the intermediate product is flat. This reaction is reversible. B) The best analogs for inhibiting the reaction are planar since these most closely resemble the flat transition state intermediate.

Beta-galactosidase splits ONPG giving a yellow color and X-gal to release a blue dye.

Some inhibitors react with the enzyme and inactivate it permanently.

distort the substrate in the required direction and help the reaction along. Consequently, some of the best competitive inhibitors are molecules that mimic the reaction intermediate. These are sometimes known as "transition state analogs." Proline race-mase inter-converts the L- and D- isomers of proline by removing an H-atom and replacing it in a different configuration (Fig. 7.35). The substrate and product are both tetrahedral about the a-carbon but the transition state is planar. The best competitive inhibitors are flat ring compounds rather than ones that look most like the substrate (Fig. 7.35).

The enzyme b-galactosidase splits many molecules in which galactose is linked to another molecule (refer to Fig. 7.27, above). To take advantage of this, researchers can use a substance called ONPG (oriAo-nitrophenyl galactoside), which consists of ortho-nitrophenol linked to galactose. When ONPG is split, the result is galactose, which is colorless, and ortho-nitrophenol, which is bright yellow (Fig. 7.36). Using ONPG allows researchers to monitor the level of b-galactosidase by measuring the appearance of the yellow color. Similarly, X-gal is split by b-galactosidase into a blue dye plus galactose (see Ch. 25 for applications). Compounds that are themselves colorless (or only pale) but react to release strongly colored products are known as chromogenic substrates.

Irreversible inhibition occurs when an inhibitor covalently modifies an enzyme, usually at the active site. Covalent inhibitors are not always analogs of the substrate. Some irreversible inhibitors react with components of the active site, other inhibitors react with other regions of the protein and may affect protein conformation, solubility or other properties. For example, the nerve agent, sarin, used in chemical warfare covalently inhibits enzymes that have serine in the active site (Fig. 7.37). The nerve agent reacts with the serine and blocks the active site permanently, inactivating the enzyme. Many hydrolytic enzymes, including proteases, rely on active serine residues. So does acetylcholine esterase, the enzyme that splits the neurotransmitter acetyl-choline. Inhibition of this causes paralysis of neuromuscular junctions and ultimately death from respiratory failure.

chromogenic substrate Colorless or pale substrate that is converted to a strongly colored product by an enzyme irreversible inhibition Type of inhibition in which an enzyme is permanently inactivated by a chemical change oriAo-nitrophenyl galactoside (ONPG) Artificial substrate for b-galactosidase that yields a yellow color upon cleavage transition state analog Enzyme inhibitor that mimics the reaction intermediate or transition state, rather than the substrate X-gal Artificial substrate for b-galactosidase that yields a blue color upon cleavage

FIGURE 7.36 b-Galactosidase Splits ONPG and X-Gal

p-Galactosidase is an enzyme that splits off galactose from other molecules to which it is attached. A) The substrate, ONPG, is used in molecular biology to measure the level of p-galactosidase activity since the reaction product, ortho-nitrophenol, is yellow and can be easily measured. B) X-Gal is another chromogenic substrate for p-galactosidase that is split releasing galactose and a fragment that reacts with oxygen in air yielding a blue dye.

FIGURE 7.37 Covalent Inhibition of Serine Enzyme by Sarin

The "nerve gas" sarin interacts with serine residues in the active site of proteins. It blocks the active site and prevents entry of the true substrate into the active site.

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