科研之友
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摘要
Angiotensin (Ang) 1-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, Arg(500), is substituted with Gln. The Arg(1098) to Lys substitution reduced Cl- binding affinity by similar to 100-fold. In the absence of Cl-, substrate binding affinity (1/K-m) of and catalytic, efficiency (k(cat)/K-m) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg(1098) to Gln substitution, and are similar (<2-foId difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl-]. The Arg(1098) 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(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 1. Since Cl- concentrations are saturating under physiological conditions and Arg(1098) 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.
- 出版日期2001-9-7
