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Article
Kinetic, Spectroscopic, and X-ray Crystallographic Characterization of the Functional E151H Aminopeptidase from Aeromonas proteolytica
Biochemistry
  • Krzysztof P. Bzymek, Utah State University
  • Aaron Moulin, Brandeis University
  • Sabina I. Swierczek, Utah State University
  • Dagmar Ringe, Brandeis University
  • Gregory A. Petsko, Brandeis University
  • Brian Bennett, Marquette University
  • Richard C. Holz, Marquette University
Document Type
Article
Language
eng
Format of Original
11 p.
Publication Date
9-1-2005
Publisher
American Chemical Society
Original Item ID
doi: 10.1021/bi0505823
Disciplines
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 kcat value of 2.0 min-1, which is over 2000 times slower than r AAP (4380 min-1). ITC experiments revealed that ZnII binds 330 and 3 times more weakly to E151H-AAP compared to r-AAP. UV−vis and EPR spectra of CoII-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 kcat and Km 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 ZnII 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

Biochemistry, Vol. 44, No. 36 (September 2005): 12030-12040. DOI.

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.

Citation Information
Krzysztof P. Bzymek, Aaron Moulin, Sabina I. Swierczek, Dagmar Ringe, et al.. "Kinetic, Spectroscopic, and X-ray Crystallographic Characterization of the Functional E151H Aminopeptidase from Aeromonas proteolytica" Biochemistry (2005) ISSN: 0006-2960
Available at: http://works.bepress.com/richard_holz/86/