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

Insulin-regulated aminopeptidase (IRAP) is a type II integral membrane prot ein belonging to the gluzincin family of metallopeptidases identified by th e characteristic Zn2+-coordination sequence element, HEXXH-(18-64X)-E. A se cond conserved sequence element, the GXMEN motif, positioned 22-32 amino ac ids N-terminal to the Zn2+-coordination sequence element distinguishes the gluzincin aminopeptidases from other gluzincins. To investigate the importa nce of the G428AMEN and H464ELAH-(18X)-E487 motifs for the activity of IRAP , mutational analysis was carried out. cDNA encoding the full-length transm embrane form of human IRAP was expressed in HEK293 cells and recombinant wi ld-type IRAP was shown to have biochemical and enzymatic properties similar to those reported for native IRAP and the soluble serum form of IRAP. Muta tional analysis using single amino-acid substitutions in the GAMEN motif (G 428A, A429G, M430K, M430E, M430I, E431D and E431A) and in the Zn2+-binding motif (H464Y. E465D, E465Q, H468Y, E487D and E487Q) resulted in decreased o r abolished aminopeptidase activity towards the leucine-para-nitroanilide s ubstrate. The results show that conservation of residues within the GAMEN a nd Zn2+-binding motifs is important for IRAP enzyme activity.