Contribution of molecular modeling and site-directed mutagenesis to the identification of a new residue, glutamate 215, involved in the exopeptidase specificity of aminopeptidase A.
Aminopeptidase A is a zinc metalloenzyme that generates brain angiotensin III, which exerts a tonic stimulatory action on blood pressure in hypertensive animals. We have previously constructed a three-dimensional model of the ectodomain of this enzyme, using the crystal structure of leukotriene A4 hydrolase/aminopeptidase as a template. According to this model, Glu-215, which is located in the active site, hydrogen bonds to the amino moiety of the inhibitor, 4-amino-4-phosphonobutyric acid (GluPhos), a phosphonic acid anologue of glutamic acid. Replacement of this residue with an aspartate or an alanine in the model abolished this interaction and led to a change in the position of the inhibitor in the active site. Mutagenic replacement of Glu-215 with an aspartate or an alanine drastically reduced the affinity of the recombinant enzymes for the substrate by a factor of 10 or 17, respectively, and the rate of hydrolysis by a factor of 14 or 6, respectively. Two isomers of GluPhos with different N-terminal amine positions differed considerably in their ability to inhibit the wild type (by a factor of 40), but not the mutated enzymes. These results, together with the interaction predicted by the model, demonstrate that Glu-215 interacts with the N-terminal amine of the substrate, thereby contributing, together with Glu-352, to the determination of the exopeptidase specificity of aminopeptidase A.
Rozenfeld
Raphaël
R
INSERM Unité 36, Collège de France, 11, place Marcelin Berthelot, 75005 Paris, France.
Iturrioz
Xavier
X
Okada
Mayumi
M
Maigret
Bernard
B
Llorens-Cortes
Catherine
C
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation / Q000235:genetics; D020689:Exopeptidases / Q000737:chemistry* / Q000235:genetics; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D043384:Glutamyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000138:chemical synthesis / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000235:genetics; D014162:Transfection
D005971:Glutamates; D063065:Organophosphonates; D011994:Recombinant Proteins; C111969:glutamate phosphonic acid; C067365:glutamate thiol; D018698:Glutamic Acid; D006639:Histidine; D020689:Exopeptidases; D043384:Glutamyl Aminopeptidase