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Arg1098 is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity

Liu, Xifu, Fernandez, Marian, Wouters, Merridee A., Heyberger, Sophie and Husain, Ahsan 2001, Arg1098 is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity, Journal of biological chemistry, vol. 276, no. 36, pp. 33518-33525, doi: 10.1074/jbc.M101495200.

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Title Arg1098 is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity
Formatted title Arg1098 is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity
Author(s) Liu, Xifu
Fernandez, Marian
Wouters, Merridee A.
Heyberger, Sophie
Husain, Ahsan
Journal name Journal of biological chemistry
Volume number 276
Issue number 36
Start page 33518
End page 33525
Total pages 8
Publisher American Society for Biochemistry and Molecular Biology
Place of publication Bethesda, Md.
Publication date 2001-09-07
ISSN 0021-9258
1083-351X
Keyword(s) catalysis
enzymes
hydrolysis
physiology
Summary 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/K m) of and catalytic efficiency (k cat/K m) 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) Arg1098 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.
Language eng
DOI 10.1074/jbc.M101495200
Field of Research 060199 Biochemistry and Cell Biology not elsewhere classified
Socio Economic Objective 970106 Expanding Knowledge in the Biological Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
Copyright notice ©2001, American Society for Biochemistry and Molecular Biology
Persistent URL http://hdl.handle.net/10536/DRO/DU:30038983

Document type: Journal Article
Collection: School of Life and Environmental Sciences
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