The catalytic role of glutamate 151 in the leucine aminopeptidase from Aeromonas proteolytica.
Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (K(m)) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants k(cat) and K(m) revealed a change in the pK(a) of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pK(a) values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base.
Bzymek
Krzysztof P
KP
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Holz
Richard C
RC
D000333:Aeromonas / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D003903:Deuterium; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008670:Metals; D011522:Protons; D011994:Recombinant Proteins; D018698:Glutamic Acid; D003903:Deuterium; D000626:Aminopeptidases; D007931:Leucyl Aminopeptidase; C038222:bacterial leucyl aminopeptidase