Structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase.

TitleStructural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase.
Publication TypeJournal Article
Year of Publication2008
AuthorsKiss, A. L., A. Palló, G. Náray-Szabó, V. Harmat, and L. Polgár
JournalJ Struct Biol
Volume162
Issue2
Pagination312-23
Date Published2008 May
ISSN1095-8657
KeywordsAmino Acid Motifs, Amino Acid Substitution, Anions, Binding Sites, Catalysis, Crystallography, X-Ray, Hydrogen Bonding, Kinetics, Peptide Hydrolases, Serine Endopeptidases
Abstract

It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(k(cat)/K(m)) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme-substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme.

DOI10.1016/j.jsb.2008.01.012
Alternate JournalJ. Struct. Biol.
PubMed ID18325786