STRUCTURAL CONSTRAINTS OF INHIBITORS FOR BINDING AT 2 ACTIVE-SITES ONSOMATIC ANGIOTENSIN-CONVERTING ENZYME

Angiotensin converting enzyme active sites from rat plasma, lung, kidn ey and testis were assessed by comparative radioligand binding studies under physiological chloride conditions. Displacement of [I-125]Ro 31 -8472 from somatic and plasma angiotensin converting enzyme by angiote nsin converting enzyme inhibitors of different structure indicated two binding sites (perindoprilat: high affinity carboxyl site, K-DC 18 +/ - 6 pM), and a single high affinity binding site on testis angiotensin converting enzyme (K-DC 20 +/- 1 pM). Displacement of [I-125]351A fro m plasma, somatic and testis angiotensin converting enzyme occurred at a single high affinity binding site. Reduction in affinity at the ami no binding site of somatic angiotensin converting enzyme was related t o an increased side chain size (lung K-DA (pM): Ro 31-8472 175 +/- 38, lisinopril 2205 +/- 1832, and 351A 2271 +/- 489), or hydrophobicity o f the competing unlabelled angiotensin converting enzyme inhibitor (lu ng K-DA (pM): quinaprilat 1267 +/- 629 and perindoprilat 824 +/- 6). T his trend was reversed at the carboxyl binding site of plasma, somatic and testis angiotensin converting enzyme. Bradykinin hydrolysis by lu ng angiotensin converting enzyme was inhibited in a similar manner by cilazaprilat or quinaprilat (F = 0.64, F-test based on the extra sum-o f-squares principle; P > 0.05), indicating the angiotensin converting enzyme carboxyl active site predominates in bradykinin cleavage. The d ata demonstrate that the two binding sites on native plasma and somati c angiotensin converting enzyme are of potentially different functiona l and structural nature, suggesting they may have different substrate specificities.