Active site of epoxide hydrolases revisited: A noncanonical residue in potato StEH1 promotes both formation and breakdown of the alkylenzyme intermediate
2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 9, 2466-2479 p.Article in journal (Refereed) Published
The carboxylate of Glu(35) in the active site of potato epoxide hydrolase StEH1 interacts with the catalytic water molecule and is the first link in a chain of hydrogen bonds connecting the active site with bulk solvent. To probe its importance to catalysis, the carboxylate was replaced with an amide through an E35Q mutation. Comparing enzyme activities using the two trans-stilbene oxide (TSO) enantiomers as substrates revealed the reaction with R,R-TSO to be the one more severely affected by the E35Q mutation, as judged by determined kinetic parameters describing the pre-steady states or the steady states of the catalyzed reactions. The hydrolysis of S,S-TSO afforded by the E35Q mutant was comparable with that of the wild-type enzyme, with only a minor decrease in activity, or a change in pH dependencies of k(cat), and the rate of alkylenzyme hydrolysis, k(3). The pH dependence of E35Q-catalyzed hydrolysis of R,R-TSO, however, exhibited an inverted titration curve as compared to that of the wild-type enzyme, with a minimal catalytic rate at pH values where the wild-type enzyme exhibited maximum rates. To simulate the pH dependence of the E35Q mutant, a shift in the acidity of the alkylenzyme had to be invoked. The proposed decrease in the pK(a) of His(300) in the E35Q mutant was supported by computer simulations of the active site electrostatics. Hence, Glu(35) participates in activation of the Asp nucleophile, presumably by facilitating channeling of protons out of the active site, and during the hydrolysis half-reaction by orienting the catalytic water for optimal hydrogen bonding, to fine-tune the acid-base characteristics of the general base His(300).
Place, publisher, year, edition, pages
2007. Vol. 46, no 9, 2466-2479 p.
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-97211DOI: 10.1021/bi062052sISI: 000244468000020PubMedID: 17284015OAI: oai:DiVA.org:uu-97211DiVA: diva2:172045