The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica

Authors

Krzysztof P. Bzymek, Utah State University
Richard C. Holz, Marquette UniversityFollow

Document Type

Article

Publication Date

7-23-2004

Source Publication

Journal of Biological Chemistry

Source ISSN

0021-9258

Abstract

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.

Comments

Published version. Journal of Biological Chemistry, Vol. 279, No. 30 (July 23, 2004): 31018-31025. DOI. © 2005 American Society for Biochemistry and Molecular Biology. Used with permission.

Richard Holz was affiliated with the Utah State University at the time of publication.

Recommended Citation

Bzymek, Krzysztof P. and Holz, Richard C., "The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica" (2004). Chemistry Faculty Research and Publications. 339.
https://epublications.marquette.edu/chem_fac/339