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Siying Ye, Siew Yeen Chai, Rebecca A. Lew, Anthony L. Albiston

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

Keywords: aminopeptidase, angiotensin IV, AT4 receptor, IRAP, memory

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 ?-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.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 388, 04/2007
Pages: 399 - 403

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