The kinetics of carboxypeptidase B activity. III. Effects of alcohol on the peptidase and esterase activities; kinetic models.

Certain of the kinetic properties of carboxypeptidase B have been described in the previous papers of this series (1, 2). The experimental constants, K, and ko,l were determined with a number of substrates, and the enzyme-inhibitor dissociation constants were evaluated with several competitive inhibitors. The present studies were undertaken in an effort to obtain knowledge of the affinity of carboxypeptidase B for its substrates through measurement of K, values, the constants for the dissociation of enzyme-substrate complexes. Slater has demonstrated that values for K, may be obtained in systems where ko, the ra,te constant for breakdown of enzymesubstrate complex to products, is varied independently of K, (3). Main (4) interpreted the alcohol-promoted activation and reversible inhibition of hydrolysis of certain nitrophenyl carboxylic acid esters by human serum cholinesterase to be examples of this type of phenomenon. On the basis of this assumption, he assigned K, values calculated from the common point of intersection obtained by extrapoIations in Lineweaver-Burk plots (5) of activities at a number of alcohol concentrations. With the hope of obtaining simiiar data from which kinetic constants could be derived for carboxypeptidase B, the peptidase and esterase activities of this enzyme were examined in the presence of low concentrations of butanol and other alcohols. The results of some of these studies were incompatible with changes in ko independent of K, and prompted a reinvestigation of the assumptions and derivations used in the determination of K,. It became evident that more than one model was compatible with the data for both peptide and ester hydrolysis. Under these circumstances, the assignment of K, values is justified only if additional experimental evidence for one model in preference to others is avaiIabIe.