Angiotensin (Ang) I-converting enzyme (ACE) is a Zn2؉ metalloprotease with two homologous catalytic domains. Both the N-and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl ؊ , but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl ؊ dependence. Angiotensin I (Ang I) 1 -converting enzyme (ACE, EC 3.4.15.1) belongs to the gluzincin family (clan MA) of metalloproteases and is a peptidyl dipeptidase with broad substrate specificity (1). Peptide hydrolysis is activated by monovalent anions such as Cl Ϫ ; this feature is unique among metalloproteases (2). The somatic form of human ACE has two homologous catalytic domains. These N-and C-domains most likely are the result of an ancient gene duplication event that occurred during vertebrate evolution (3, 4). Invertebrate ACE has a single, Cl Ϫ -sensitive catalytic domain (5). The physiological substrates of ACE include Ang I, bradykinin, substance P, and AcSDKP (2); however, there are significant differences between the catalytic domains in catalytic efficiencies for the hydrolysis of these substrates (6, 7).Cl Ϫ dependence of hydrolysis is substrate-specific. For example, the degree of enzyme activation by Cl Ϫ and its apparent dissociation constant (K d,app ) associated with this activation is high for Ang I and low for bradykinin (8, 9). To define the molecular determinants in the substrate structure responsible for these differences, Riordan and colleagues (8, 9) studied the hydrolysis of a collection of tripeptide substrates with varying C-terminal dipeptide structures. They observed that a P 1 Ј-or P 2 Ј-(Arg/Lys) 2 in the tripeptide substrate was necessary and sufficient for high affinity Cl Ϫ binding. Bradykinin, like these substrates, but unlike Ang I, contains a basic residue in its C-terminal dipeptide. Also, hydrolytic activity of ACE for tripeptide substrates with a P 1 Ј-or P 2 Ј-(Arg/Lys) is less affected by Cl Ϫ than for those that lack a basic P 1 Ј or P 2 Ј residue. Because chemical modification of ACE that leads to methylation of Lys residues has selective effects on hydrolytic activity for furanacryloyl-FGG-COO Ϫ (inactivated by Ͼ99%) and furanacryloyl-FFR-COO Ϫ (activity reduced by ϳ50%) it was proposed that a critical Lys is part of the Cl Ϫ -binding site of ACE (10). The study by Shapiro and Riordan (10) had provided only circumstantial evidence that a critical Lys was involved in Cl Ϫ binding, and in principle it is best to consider both types of basic residues as possibilities. To identify the Cl Ϫ -binding residue(s) in ACE we made single substitutions with Gln of all conserved arginines or lysines in the human ACE C-domain. Here we show that the primary site of Cl Ϫ binding in the C-domain is Arg 1098 irrespective of whether the substrate is the decapeptide Ang I or a short peptide with or without a basic residue a...

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N-and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His 1089 as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His 1089 to Ala or Leu. At pH 7.5, with Ang I as substrate, k 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-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His 1089 3 Ala mutation. ACE C-domain-dependent 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 alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins 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 of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.

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