A tyrosine residue essential for catalytic activity in aminopeptidase A.
Aminopeptidase A (EC 3.4.11.7; APA) is a 130 kDa membrane-bound zinc enzyme that contains the consensus sequence HEXXH (residues 385-389) conserved among the zinc metalloprotease family. In this motif, both histidine residues and the glutamic residue were shown to be involved respectively in zinc co-ordination and catalytic activity. Treatment of APA with N-acetylimidazole results in a loss of enzymic activity; this is prevented by the competitive aminopeptidase inhibitor amastatin, suggesting the presence of an important tyrosine, lysine or cysteine residue at the active site of APA. A tyrosine residue was previously proposed to be involved in the enzymic activity of aminopeptidase N. Furthermore sequence alignment of mouse APA with other monozinc aminopeptidases indicates the presence of a conserved tyrosine (Tyr-471 in APA). The functional role of Tyr-471 in APA was investigated by replacing this residue with a phenylalanine (Phe-471) or a histidine (His-471) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of both mutants were similar to that of the wild-type enzyme, whereas kcat values were decreased by three orders of magnitude and corresponded to a variation in free energy of the rate-limiting step by 4.0 and 4.2 kcal/mol (0.96 and 1.00 kJ/mol) for the Phe-471 and His-471 mutants respectively. The mutation did not modify the inhibitory potency of a thiol-containing inhibitor that strongly chelates the active-site zinc ion, whereas that of a putative analogue of the transition state presumed to mimic the reaction intermediate was reduced. Taken together, these results strongly suggest that the Tyr-471 hydroxy group participates in catalysis by stabilizing the transition state complex through interaction with the oxyanion.
Vazeux
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INSERM Unit 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France.
Iturrioz
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X
Corvol
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Llorens-Cortès
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C
D019943:Amino Acid Substitution; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005971:Glutamates / Q000494:pharmacology*; D043384:Glutamyl Aminopeptidase; D006639:Histidine / Q000235:genetics; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000494:pharmacology*; D010649:Phenylalanine / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005971:Glutamates; D007093:Imidazoles; D063065:Organophosphonates; D011994:Recombinant Proteins; C111969:glutamate phosphonic acid; C067365:glutamate thiol; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; D008666:Metalloendopeptidases; C012160:N-acetylimidazole