ANGIOTENSIN-CONVERTING ENZYME PREFERENTIALLY HYDROLYZES TRANS ISOMER OF PROLINE-CONTAINING SUBSTRATE

An analysis of the hydrolysis kinetics of the synthetic angiotensin-co nverting enzyme (ACE) substrate benzoyl-phenylalanyl-alanyl-proline (B PAP) in the intact lung suggested that 12-15% of the BPAP was in a for m that could not be hydrolyzed by ACE in the time course of a single p ass through the lungs [C. A. Dawson et al. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H853-H865, 1989]. BPAP has been found to exist as a mixture of cis and trans isomers in a ratio of approximately 14:86 i n aqueous solution at equilibrium. Thus, one possible explanation for the incomplete hydrolysis of BPAP on passage through the intact lung i s that the trans form is the preferred substrate for ACE. To examine t his hypothesis, we measured BPAP hydrolysis by ACE in vitro over a ran ge of ACE concentrations and in the presence and absence of the peptid yl-prolyl cis-trans isomerase cyclophilin. In the presence of a suffic ient concentration of ACE and in the absence of cyclophilin, hydrolysi s of [H-3]BPAP by ACE followed biexponential progress curves, consiste nt with the hypothesis that the rate of hydrolysis of the majority (ap proximately 87%) of the substrate is proportional to ACE concentration , whereas the hydrolysis rate of the remaining substrate fraction is i ndependent of enzyme concentration. The addition of cyclophilin result ed in an increase in the ACE-independent rate constant, an effect that was reversed by the cyclophilin inhibitor cyclosporin A. These result s suggest that the enzyme-independent rate constant represents the rat e of cis-trans isomerization and that the enzyme-dependent rate consta nt represents the hydrolysis of the trans isomer. Thus, these data are consistent with the hypothesis that ACE preferentially hydrolyzes the trans isomer of BPAP.