Mutational analysis of the active site of human insulin‐regulated aminopeptidase

Abstract

Insulin‐regulated aminopeptidase (IRAP) is a type II integral membrane protein belonging to the gluzincin family of metallopeptidases identified by the characteristic Zn²⁺‐coordination sequence element, HEXXH‐(18–64X)‐E. A second conserved sequence element, the GXMEN motif, positioned 22–32 amino acids N‐terminal to the Zn²⁺‐coordination sequence element distinguishes the gluzincin aminopeptidases from other gluzincins. To investigate the importance of the G428AMEN and H464ELAH‐(18X)‐E487 motifs for the activity of IRAP, mutational analysis was carried out. cDNA encoding the full‐length transmembrane form of human IRAP was expressed in HEK293 cells and recombinant wild‐type IRAP was shown to have biochemical and enzymatic properties similar to those reported for native IRAP and the soluble serum form of IRAP. Mutational analysis using single amino‐acid substitutions in the GAMEN motif (G428A, A429G, M430K, M430E, M430I, E431D and E431A) and in the Zn²⁺‐binding motif (H464Y, E465D, E465Q, H468Y, E487D and E487Q) resulted in decreased or abolished aminopeptidase activity towards the leucine‐para‐nitroanilide substrate. The results show that conservation of residues within the GAMEN and Zn²⁺‐binding motifs is important for IRAP enzyme activity.