Arg(1098) is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity

Angiotensin (Ang) 1-converting enzyme (ACE) is a Zn2+ metalloprotease with two homologous catalytic domains. Both the N- and C-terminal domains are pe ptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I i s increased by Cl-, but the molecular mechanism of this regulation is uncle ar. A search for single substitutions to Gln among all conserved basic resi dues (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(500), is substituted with Gln. The Arg(1098) to L ys substitution reduced Cl- binding affinity by similar to 100-fold. In the absence of Cl-, substrate binding affinity (1/K-m) of and catalytic, effic iency (k(cat)/K-m) for Ang I hydrolysis are increased 6.9- and 32-fold, res pectively, by the Arg(1098) to Gln substitution, and are similar (<2-foId d ifference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl-]. The Arg(1098) to Gln substitution also eliminate s Cl- dependence for hydrolysis of tetrapeptide substrates, but activity to ward these substrates is similar to that of the Wildtype C-domain in the ab sence of Cl-. These findings indicate that: 1) Arg(1098) is a critical resi due of the C-domain Cl--binding site and 2) a basic side chain is necessary for Cl- dependence. For tetrapeptide substrates, the inability of R1098Q t o recreate the high affinity state generated by the Cl-C-domain interaction suggests that substrate interactions with the enzyme-bound Cl- are much mo re important for the hydrolysis of short substrates than for Ang 1. Since C l- concentrations are saturating under physiological conditions and Arg(109 8) 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 all ow cleavage of short cognate peptide substrates at high catalytic efficienc ies.