Macrocyclic Enzyme Model Systems. Catalytic Activity of Cyclic Peptides Involving Hydrophobic Segments

Abstract

Abstract In order to develop an effective hydrolase model, BCP-1 was synthesized and its kinetic behavior investigated. The corresponding linear hexapeptide was prepared by the solid phase method and subjected to cyclization by the azide method. BCP-1 enhanced the hydrolysis of p-nitrophenyl carboxylates bearing a long alkyl chain. The pH-rate profile for the hydrolysis of p-nitrophenyl hexadecanoate (PNPP) as effected by BCP-1 was not of a bell-shaped type but a sigmoid, from which the kinetic pKa value was estimated as 12.3. The initial reaction rate was found to level off beyond a certain range of substrate concentration (saturation-type kinetics), the kinetic data being analyzed on the basis of Michaelis-Menten scheme. The large hydrophobic binding ability of BCP-1 toward PNPP (Km(app) \simeq10−6M) was attributed to its double-layered bicyclic structure. The Hammett plot for the hydrolysis of substituted phenyl hexadecanoates gave a satisfactory linear correlation. Consequently, the acyl transfer from the bound substrate to the imidazole anion of the histidyl residue of BCP-1 was referred to the rate-determining step. The large negative entropy change for the acyl transfer step seems to indicate that the Michaelis complex does not have a conformation favorable for such acyl transfer process. The catalytic effectiveness (kcat⁄Km(app)) for the BCP-1 catalysis in PNPP hydrolysis is comparable to that for enzymatic hydrolysis.