A THIOAMIDE SUBSTRATE OF CARBOXYPEPTIDASE-A

Carbobenzoxythioglycyl-L-phenylalanine [CbzNHCH2C (.dbd.S) Phe, Z-GlyS-Phe] was synthesized as a thioamide analog of Z-Gly-Phe, a known substrate of [bovine] carboxypeptidase A (CPA). By use of a ninhydrin-based assay and Z-Gly-Gly-Phe as the substrate, Z-GlyS-Phe was shown to be a weak, competitive inhibitor of CPA (Ki = 1.4 mM). The L isomer (but not the D) of Z-GlyS-Phe proved to be a substrate for CPA (Km = 1.1 mM and kcat = 5.3 s-1 at pH 7.5), binding with comparable affinity to, but hydrolyzing at 10% the rate of, the oxo analog Z-Gly-Phe. The CPA-catalyzed hydrolysis of Z-GlyS-Phe involves only C.sbd.N bond cleavage, give carbobenzoxythioglycine and phenylalanine.