Identification of glutamate residues essential for catalytic activity and zinc coordination in aminopeptidase A.
Aminopeptidase A (EC 3.4.11.7, APA) is a homodimeric membrane-bound glycoprotein that contains the consensus sequence HEXXH(385-389) found in zinc metallopeptidases such as thermolysin. The x-ray structure of the latter enzyme revealed that the two histidines of this motif are two of the three zinc-coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis. Alignment of the sequence of mouse APA with those of the already characterized metallopeptidases showed the presence of several conserved amino acids such as a glutamate residue in position 408 which may constitute the putative third zinc ligand. The functional implication of this residue and the role of glutamate 386 in the HELVH(385-389) motif of APA have been investigated by replacing these residues with an aspartate (Asp-386, Asp-408) or an alanine (Ala-386, Ala-408) by site-directed mutagenesis. Expressed mutated proteins in position 386 showed no APA activity. Ala-408 was also inactive, and Asp-408 had 5% of the wild type enzyme activity and a similar Km. 65Zn incorporation measurements indicated that Ala-386 binds the zinc ion as well as the wild type enzyme, whereas the Ala-408 mutant did not. These results provide evidence that Glu-408 is the third zinc-coordinating residue of APA, confirm the presumed involvement of Glu-386 in the catalytic process of the enzyme, and identify APA as a zinc metallopeptidase functionally similar to thermolysin.
Vazeux
G
G
INSERM Unit 36, Collège de France, 3, rue d'Ulm, 75005 Paris, France.
Wang
J
J
Corvol
P
P
Llorens-Cortès
C
C
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D005971:Glutamates / Q000737:chemistry; D043384:Glutamyl Aminopeptidase; D008562:Membrane Glycoproteins / Q000737:chemistry; D008666:Metalloendopeptidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013820:Thermolysin / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D017931:DNA Primers; D005971:Glutamates; D008562:Membrane Glycoproteins; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; D008666:Metalloendopeptidases; D013820:Thermolysin; D015032:Zinc