CHLOROKETONE HYDROLYSIS BY CHYMOTRYPSIN AND N-METHYLHISTIDYL-57-CHYMOTRYPSIN - IMPLICATIONS FOR THE MECHANISM OF CHYMOTRYPSIN INACTIVATION BY CHLOROKETONES

We have examined the reaction of N-(benzyloxycarbonyl)-L-alanyl- L-gly cyl-L-phenylalanyl chloromethyl ketone (ZAGFCMK) with chymotrypsin (Ch t) and have found that, in addition to irreversible alkylation of the enzyme, some of the corresponding hydroxymethyl ketone is produced. Fo r each molecule of hydroxy ketone formed, 3.6 molecules of chymotrypsi n are inactivated. Chloroketone hydrolysis is also observed with chymo trypsin methylated at N-3 of the active site histidine (MeCht). The hy drolysis proceeds slowly (k = 0.14 min(-1)). Alkylation of the modifie d enzyme was not observed. An initial burst of free chloride is detect ed during the MeCht-catalyzed hydrolysis. The magnitude of the chlorid e burst is proportional to the enzyme concentration in an approximate 1:1 stoichiometry and indicates a relatively rapid chloride-releasing step which gives rise to an intermediate which is more slowly converte d to hydroxy ketone. We have also investigated both the solution and M eCht-mediated hydrolysis of the S isomer of N-acetyl-L-alanyl-L-phenyl alanyl chloroethyl ketone (S-AcAFCEK). We have concluded that the none nzymatic hydrolysis proceeds with inversion of configuration at the st ereocenter, while the enzymatic process occurs with retention of confi guration. The two nucleophilic displacements attending the MeCht-media ted hydrolysis of S-AcAFCEk imply the formation of an intermediate, po ssibly of an epoxy ether, formed by internal displacement of the chlor ide by the oxyanion of the initially generated enzyme-chloroketone hem iketal adduct.