Insulin-regulated aminopeptidase : analysis of peptide substrate and inhibitor binding to the catalytic domain

Ye, Siying, Chai, Siew Yeen, Lew, Rebecca A. and Albiston, Anthony L. 2007, Insulin-regulated aminopeptidase : analysis of peptide substrate and inhibitor binding to the catalytic domain, Biological chemistry, vol. 388, no. 4, pp. 399-403, doi: 10.1515/BC.2007.044.

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Title Insulin-regulated aminopeptidase : analysis of peptide substrate and inhibitor binding to the catalytic domain
Author(s) Ye, SiyingORCID iD for Ye, Siying
Chai, Siew Yeen
Lew, Rebecca A.
Albiston, Anthony L.
Journal name Biological chemistry
Volume number 388
Issue number 4
Start page 399
End page 403
Publisher Walter de Gruyter GmbH & Co. KG
Place of publication Berlin, Germany
Publication date 2007-03
ISSN 1431-6730
Keyword(s) aminopeptidase
angiotensin IV
AT4 receptor
Summary Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine β-naphthalamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.
Language eng
DOI 10.1515/BC.2007.044
Field of Research 119999 Medical and Health Sciences not elsewhere classified
Socio Economic Objective 970111 Expanding Knowledge in the Medical and Health Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
Copyright notice ©2007, Walter de Gruyter
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Document type: Journal Article
Collections: Faculty of Health
School of Medicine
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