Kinetic, Spectroscopic, and X-ray Crystallographic Characterization of the Functional E151H Aminopeptidase from Aeromonas proteolytica

Authors

Krzysztof P. Bzymek, Utah State University
Aaron Moulin, Brandeis University
Sabina I. Swierczek, Utah State UniversityFollow
Dagmar Ringe, Brandeis University
Gregory A. Petsko, Brandeis University
Brian Bennett, Marquette UniversityFollow
Richard C. Holz, Marquette UniversityFollow

Document Type

Article

Language

eng

Format of Original

11 p.

Publication Date

9-2005

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

doi: 10.1021/bi0505823

Abstract

Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the d-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in DppA. Substitution of E151 with histidine resulted in an active AAP enzyme exhibiting a k_cat value of 2.0 min^-1, which is over 2000 times slower than r AAP (4380 min^-1). ITC experiments revealed that Zn^II binds 330 and 3 times more weakly to E151H-AAP compared to r-AAP. UV−vis and EPR spectra of Co^II-loaded E151H-AAP indicated that the first metal ion resides in a hexacoordinate/pentacoordinate equilibrium environment, whereas the second metal ion is six-coordinate. pH dependence of the kinetic parameters k_cat and K_m for the hydrolysis of l-leucine p-nitroanilide (l-pNA) revealed a change in an ionization constant in the enzyme−substrate complex from 5.3 in r-AAP to 6.4 in E151H-AAP, consistent with E151 in AAP being the active-site general acid/base. Proton inventory studies at pH 8.50 indicate the transfer of one proton in the rate-limiting step of the reaction. Moreover, the X-ray crystal structure of [ZnZn(E151H-AAP)] has been solved to 1.9 Å resolution, and alteration of E151 to histidine does not introduce any major conformational changes to the overall protein structure or the dinuclear Zn^II active site. Therefore, a histidine residue can function as the general acid/base in hydrolysis reactions of peptides and, through analogy of the role of E151 in AAP, H115 in DppA likely shuttles a proton to the leaving group of the substrate.

Comments

Accepted version. Biochemistry, Vol. 44, No. 36 (September 2005): 12030-12040. DOI. © 2005 American Chemical Society Publications. Used with permission.

Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.

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

Recommended Citation

Bzymek, Krzysztof P.; Moulin, Aaron; Swierczek, Sabina I.; Ringe, Dagmar; Petsko, Gregory A.; Bennett, Brian; and Holz, Richard C., "Kinetic, Spectroscopic, and X-ray Crystallographic Characterization of the Functional E151H Aminopeptidase from Aeromonas proteolytica" (2005). Physics Faculty Research and Publications. 35.
https://epublications.marquette.edu/physics_fac/35