Properties of analogues of an intermediate in the process of mechanism-based inactivation of carboxypeptidase A

Abstract Carboxypeptidase A (CPA), and other zinc-dependent proteases, facilitate an α deprotonation of judiciously designed ketones and amides. This adventitious reaction has been used in the development of effective mechanism-based inactivators for this family of enzymes. N -Acryloyl- l -phenylalanine, an intermediate in the process of mechanism-based inactivation of CPA by N -(3-chloropropionyl)- l -phenylalanine, was shown to be an affinity inactivator, but also a very poor substrate for the enzyme. Similarly, O -(acryloyl)- l -3-phenyllactate was shown to be both an affinity inactivator and a poor substrate for CPA. However, consistent with the trend established with other ester and amide substrates for CPA, O -(acryloyl)- l -3-phenyllactate is a better substrate than N -acryloyl- l -phenylalanine. N -(Propiolyl)- l -phenylalanine served only as a poor substrate for the enzyme. To gain insight into enzyme inactivation and the unexpected poor turnover of these molecules, molecular modeling of these compounds with the crystal structure of CPA was carried out. These analyses suggested that the smaller size of these molecules permits a binding mode which is somewhat different in the active site than with typical larger substrates, such that the transition-state species for hydrolysis is not greatly stabilized by the enzyme. The slow turnover of these species, along with their specific binding interactions with the enzyme active site have implications for the inactivation chemistry of CPA and other zinc proteases by this family of mechanism-based inactivators.