Arg Is Critical for the Chloride Dependence of Human Angiotensin I-converting Enzyme C-domain Catalytic Activity*

Angiotensin (Ang) I-converting enzyme (ACE) is a Zn metalloprotease with two homologous catalytic domains. Both the Nand 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. Cl dependence is also lost when the equivalent Arg in the N-domain, Arg, is substituted with Gln. The Arg to Lys substitution reduced Cl binding affinity by 100fold. In the absence of Cl , substrate binding affinity (1/Km) of and catalytic efficiency (kcat/Km) for Ang I hydrolysis are increased 6.9and 32-fold, respectively, by the Arg to Gln substitution, and are similar (<2-fold difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl ]. The Arg to Gln substitution also eliminates Cl dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wildtype C-domain in the absence of Cl . These findings indicate that: 1) Arg is a critical residue of the Cdomain Cl -binding site and 2) a basic side chain is necessary for Cl dependence. For tetrapeptide substrates, the inability of R1098Q to recreate the high affinity state generated by the Cl -C-domain interaction suggests that substrate interactions with the enzymebound Cl are much more important for the hydrolysis of short substrates than for Ang I. Since Cl concentrations are saturating under physiological conditions and Arg is not critical for Ang I hydrolysis, we speculate that the evolutionary pressure for the maintenance of the Cl -binding site is its ability to allow cleavage of short cognate peptide substrates at high catalytic efficiencies.