Angiotensin-converting enzyme preferentially hydrolyzes trans isomer of proline-containing substrate.
An analysis of the hydrolysis kinetics of the synthetic angiotensin-converting enzyme (ACE) substrate benzoyl-phenylalanyl-alanyl-proline (BPAP) in the intact lung suggested that 12-15% of the BPAP was in a form that could not be hydrolyzed by ACE in the time course of a single pass 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 in aqueous solution at equilibrium. Thus, one possible explanation for the incomplete hydrolysis of BPAP on passage through the intact lung is that the trans form is the preferred substrate for ACE. To examine this hypothesis, we measured BPAP hydrolysis by ACE in vitro over a range of ACE concentrations and in the presence and absence of the peptidyl-prolyl cis-trans isomerase cyclophilin. In the presence of a sufficient concentration of ACE and in the absence of cyclophilin, hydrolysis of [3H]BPAP by ACE followed biexponential progress curves, consistent with the hypothesis that the rate of hydrolysis of the majority (approximately 87%) of the substrate is proportional to ACE concentration, whereas the hydrolysis rate of the remaining substrate fraction is independent of enzyme concentration. The addition of cyclophilin resulted in an increase in the ACE-independent rate constant, an effect that was reversed by the cyclophilin inhibitor cyclosporin A. These results suggest that the enzyme-independent rate constant represents the rate of cis-trans isomerization and that the enzyme-dependent rate constant represents the hydrolysis of the trans isomer.(ABSTRACT TRUNCATED AT 250 WORDS)
Merker
M P
MP
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee 53226.
Dawson
C A
CA
Bongard
R D
RD
Roerig
D L
DL
Haworth
S T
ST
Linehan
J H
JH
D000591:Amino Acid Isomerases / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D016572:Cyclosporine / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase; D011392:Proline / Q000378:metabolism*; D011817:Rabbits; D013237:Stereoisomerism
D002352:Carrier Proteins; D009842:Oligopeptides; C029689:benzoylphenylalanyl-alanyl-proline; D016572:Cyclosporine; D011392:Proline; D007703:Peptidyl-Dipeptidase A; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase