Histidine 450 plays a critical role in catalysis and, with Ca2+, contributes to the substrate specificity of aminopeptidase A.
Aminopeptidase A (EC 3.4.11.7, APA) is a 130 kDa membrane-bound protease that contains the HEXXH consensus sequence found in the zinc metalloprotease family, the zincins. In addition to the catalytic zinc atom, APA contains a Ca2+ ion that increases its enzymatic activity. Aligning the sequences of the mouse APA, APN, and other monozinc aminopeptidases led to the identification of a conserved histidine (His 450 in mouse APA). Replacing this residue with a phenylalanine (Phe 450) by site-directed mutagenesis resulted in markedly lower levels of APA activity and in a change in the sensitivity of APA to Ca2+ (the EC50 for Ca2+ was 25 microM in the wild type and only 279 microM in the mutant). Kinetic studies, with a supramaximal Ca2+ concentration (4 mM), showed that the Km of the mutant enzyme for the substrate alpha-L-glutamyl-beta-naphthylamide was 25 times higher than that of the wild type, whereas the kcat value was much lower (factor of 22). Thus, overall, the wild-type enzyme had a cleavage efficiency that was 571 times higher than that of the mutant. The inhibitory potencies of two different classes of inhibitors, a glutamate thiol and a glutamate phosphonate compound, were significantly lower (factors of 19 and 22, respectively) for the mutated enzyme than for the wild-type enzyme. In contrast, inhibition by lysine thiol was unaffected. These data strongly suggest that His 450 is critical for catalytic activity and is involved in substrate binding via interaction with the P1 carboxylate side chain of the substrate. Furthermore, His 450, together with Ca2+, may contribute to the substrate specificity of APA for N-terminal acidic amino acid residues.
Iturrioz
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INSERM Unité 36, Collège de France, 3, rue d'Ulm, 75005 Paris, France.
Vazeux
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Célérier
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Corvol
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Llorens-Cortès
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D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000592:standards; D000818:Animals; D001216:Asparagine / Q000235:genetics; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002384:Catalysis; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005971:Glutamates / Q000494:pharmacology; D043384:Glutamyl Aminopeptidase; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism / Q000592:standards; D013379:Substrate Specificity / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005971:Glutamates; D063065:Organophosphonates; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C111969:glutamate phosphonic acid; C067365:glutamate thiol; D006639:Histidine; D001216:Asparagine; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; D008239:Lysine; D002118:Calcium