Arg1098 Is Critical for the Chloride Dependence of Human Angiotensin I-converting Enzyme C-domain Catalytic Activity

Abstract

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. Cl-dependence is also lost when the equivalent Arg in the N-domain, Arg500, is substituted with Gln. The Arg1098 to Lys substitution reduced Cl-binding affinity by ∼100-fold. In the absence of Cl-, substrate binding affinity (1/Km) of and catalytic efficiency (kcat/Km) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg1098 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 Arg1098 to Gln substitution also eliminates Cl- dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wild-type C-domain in the absence of Cl-. These findings indicate that: 1) Arg 1098 is a critical residue 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 to recreate the high affinity state generated by the Cl--C-domain interaction suggests that substrate interactions with the enzyme-bound Cl- are much more important for the hydrolysis of short substrates than for Ang I. Since Cl - concentrations are saturating under physiological conditions and Arg1098 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.