Angiotensin I-converting enzyme transition state stabilization by His(1089) - Evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin fa mily of zinc metalloproteinases that contains two homologous catalytic doma ins. Both the N- and C-terminal domains are peptidyl-dipeptidases that cata lyze Ang II formation and bradykinin degradation. Multiple sequence alignme nt was used to predict His(1089) as the catalytic residue in human ACE C-do main that, by analogy with the prototypical gluzincin, thermolysin, stabili zes the scissile carbonyl bond through a hydrogen bond during transition st ate binding. Site-directed mutagenesis was used to change His(1089) to Ala or Leu, At pH 7.5, with Ang I as substrate, F-cat/K-m values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wildtype enzyme and were mainly due to a decrease in catalytic rate (k(cat) ) with minor effects on ground state substrate binding (K-m), A 120,000-fol d decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His(1089) --> Ala mutation. ACE C-domain-depend ent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10. 5, indicating that the His(1089) side chain allows ACE to function as an al kaline peptidyl-dipeptidase. Since transition state mutants of other gluzin cins show pH optima shifts toward the alkaline, this effect of His(1089) on the ACE pH optimum and its ability to influence transition state binding o f the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.