Arg1098 is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity
Data(s) |
07/09/2001
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Resumo |
Angiotensin (Ang) I-converting enzyme (ACE) is a Zn<sup>2</sup>+ 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<sup>500</sup>, is substituted with Gln. The Arg<sup>1098</sup> to Lys substitution reduced Cl− binding affinity by ∼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<sup>1098</sup> 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<sup>1098</sup> 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<sup>1098</sup> 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 Arg<sup>1098</sup> 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. <br /> |
Identificador | |
Idioma(s) |
eng |
Publicador |
American Society for Biochemistry and Molecular Biology |
Relação |
http://dro.deakin.edu.au/eserv/DU:30038983/wouters-arg1098-2001.pdf http://dx.doi.org/10.1074/jbc.M101495200 |
Direitos |
2001, American Society for Biochemistry and Molecular Biology |
Palavras-Chave | #catalysis #enzymes #hydrolysis #physiology |
Tipo |
Journal Article |