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Angiotensin I-converting enzyme transition state stabilization by HIS1089 : evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases

Fernandez, Marian, Liu, Xifu, Wouters, Merridee A., Heyberger, Sophie and Husain, Ahsan 2001, Angiotensin I-converting enzyme transition state stabilization by HIS1089 : evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases, Journal of biological chemistry, vol. 276, no. 7, pp. 4998-5004, doi: 10.1074/jbc.M009009200.

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Title Angiotensin I-converting enzyme transition state stabilization by HIS1089 : evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases
Formatted title Angiotensin I-converting enzyme transition state stabilization by HIS1089 : evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases
Author(s) Fernandez, Marian
Liu, Xifu
Wouters, Merridee A.
Heyberger, Sophie
Husain, Ahsan
Journal name Journal of biological chemistry
Volume number 276
Issue number 7
Start page 4998
End page 5004
Total pages 7
Publisher American Society for Biochemistry and Molecular Biology
Place of publication Baltimore, Md.
Publication date 2001
ISSN 0021-9258
1083-351X
Keyword(s) angiotensin I-converting enzyme
enzyme catalysis
enzyme regulation
catalysis
degradation
enzymes
hydrogen bonds
mutagenesis
pH effects
zinc
Summary Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His1089 as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His1089 to Ala or Leu. At pH 7.5, with Ang I as substrate, kcat/Km values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (kcat) with minor effects on ground state substrate binding (Km). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His1089 --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His1089 side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His1089 on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.
Language eng
DOI 10.1074/jbc.M009009200
Field of Research 060199 Biochemistry and Cell Biology not elsewhere classified
Socio Economic Objective 970106
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:30038973

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