Mutational analysis of the active site of human insulin-regulated aminopeptidase.
Insulin-regulated aminopeptidase (IRAP) is a type II integral membrane protein belonging to the gluzincin family of metallopeptidases identified by the characteristic Zn(2+)-coordination sequence element, HEXXH-(18-64X)-E. A second conserved sequence element, the GXMEN motif, positioned 22-32 amino acids N-terminal to the Zn(2+)-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 Zn(2+)-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 Zn(2+)-binding motifs is important for IRAP enzyme activity.
Laustsen
P G
PG
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Vang
S
S
Kristensen
T
T
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D010122:Cystinyl Aminopeptidase; D004252:DNA Mutational Analysis; D006801:Humans; D017354:Point Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D015032:Zinc / Q000378:metabolism
D011994:Recombinant Proteins; D000626:Aminopeptidases; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase; D015032:Zinc