Examples of Enzyme-Catalyzed Reactions

Examples of Enzyme-Catalyzed Reactions

Chymotrypsin is an enzyme that catalyzes the hydrolysis of peptide bonds, withsome speciÞcity for residues containing aromatic side chains. Chymotrypsin also cleaves peptide bonds at other sites, such as leucine, histidine, and glutamine, but with a lower frequency than at aromatic amino acid residues. It also catalyzes the hydrolysis of ester bonds.

Although ester hydrolysis is not important to the physiological role of chy-motrypsin in the digestion of proteins, it is a convenient model system for investigating the enzymeÕs catalysis of hydrolysis reactions. The usual labora-tory procedure is to use p-nitrophenyl esters as the substrate and to monitor the progress of the reaction by the appearance of a yellow color in the reaction mixture caused by the production of p-nitrophenolate ion.

In a typical reaction in which a p-nitrophenyl ester is hydrolyzed by chymo-trypsin, the experimental rate of the reaction depends on the concentration of the substrateÑin this case, the p-nitrophenyl ester. At low substrate concen-trations, the rate of reaction increases as more substrate is added. At higher substrate concentrations, the rate of the reaction changes very little with the addition of more substrate, and a maximum rate is reached. When these results are presented in a graph, the curve is hyperbolic (Figure 6.6).

Another enzyme-catalyzed reaction is the one catalyzed by the enzyme aspar-tate transcarbamoylase (ATCase). This reaction is the first step in a pathwayleading to the formation of cytidine triphosphate (CTP) and uridine triphos-phate (UTP), which are ultimately needed for the biosynthesis of RNA and DNA. In this reaction, carbamoyl phosphate reacts with aspartate to produce carbamoyl aspartate and phosphate ion.

Reaction catalyzed by aspartate transcarbamoylase

The rate of this reaction also depends on substrate concentrationÑin this case, the concentration of aspartate (the carbamoyl phosphate concentration is kept constant). Experimental results show that, once again, the rate of the reaction depends on substrate concentration at low and moderate concentrations, and, once again, a maximum rate is reached at high substrate concentrations.

There is, however, one very important difference. For this reaction, a graph showing the dependence of reaction rate on substrate concentration has a sig-moidal rather than hyperbolic shape (Figure 6.7).

Why do chymotrypsin and ATCase have different velocity curves?

The results of experiments on the reaction kinetics of chymotrypsin and aspartate transcarbamoylase are representative of experimental results obtained with many enzymes. The overall kinetic behavior of many enzymes resembles that of chymotrypsin, while other enzymes behave similarly to aspartate transcarbamoylase. We can use this information to draw some general conclusions about the behavior of enzymes. The comparison between the kinetic behaviors of chymotrypsin and ATCase is reminiscent of the relationship between the oxygen-binding behaviors of myoglobin and hemoglobin. ATCase and hemoglobin are allosteric proteins; chymotrypsin and myoglobin are not. (Recall that allosteric proteins are the ones in which subtle changes at one site affect structure and function at another site. Cooperative effects, such as the fact that the binding of the Þrst oxygen molecule to hemoglobin makes it easier for other oxygen molecules to bind, are a hallmark of allosteric proteins.) The differences in behavior between allosteric and nonallosteric proteins can be understood in terms of models based on structural differences between the two kinds of proteins. When we encounter the mechanisms of the many enzyme-catalyzed reactions in subsequent, we shall need a model that explains the hyperbolic plot of kinetic data for nonallosteric enzymes and another model that explains the sigmoidal plot for allosteric enzymes. The Michaelis-Menten model is widely used for nonallosteric enzymes, and several models are used for allosteric enzymes.

Summary

Chymotrypsin is an enzyme that cleaves peptides near amino acids with aromatic side-chains. It can be studied by using a substrate analog con-taining p-nitrophenylacetate.

When the velocity of chymotrypsin is plotted versus its substrate, the curve is a hyperbola.

Aspartate transcarbamoylase is an enzyme that is involved in the synthesis of nucleotides.

When the velocity of aspartate transcarbamoylase is plotted versus aspar-tate, the curve is sigmoidal.

The difference between the velocity curves for chymotrypsin and aspar-tate transcarbamoylase demonstrates the difference between an allosteric enzyme and a nonallosteric enzyme.