Arg1098Is Critical for the Chloride Dependence of Human Angiotensin I-converting Enzyme C-domain Catalytic Activity

Xifu Liu, Marian Fernandez, Merridee A. Wouters, Sophie Heyberger, Ahsan Husain
2001 Journal of Biological Chemistry  
Angiotensin (Ang) I-converting enzyme (ACE) is a Zn 2؉ 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
more » ... 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 ϳ100fold. 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-fold 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 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 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. . 1 The abbreviations used are: Ang I, angiotensin I; Ang II, angiotensin II; ACE, angiotensin I-converting enzyme; HPLC, high performance liquid chromatography. 2 The nomenclature used for the individual amino acids (P 1 , P 1 Ј, etc.) of a substrate and the subsites (S 1 , S 1 Ј, etc.) of the enzyme is that of Schechter and Berger (23). Amino acid residues of substrates numbered P 1 , P 2 , etc., are towards the N-terminal direction, and P 1 Ј, P 2 Ј etc., are towards the C-terminal direction from the scissile bond.
doi:10.1074/jbc.m101495200 pmid:11432860 fatcat:6w366surfjf65chbjwsuxsz4ye