Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F16%3A00460095" target="_blank" >RIV/61388971:_____/16:00460095 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1016/j.abb.2015.12.008" target="_blank" >http://dx.doi.org/10.1016/j.abb.2015.12.008</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.abb.2015.12.008" target="_blank" >10.1016/j.abb.2015.12.008</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics

Popis výsledku v původním jazyce

A detailed kinetic study based on steady-state and pre-steady-state measurements is described for the highly enantioselective epoxide hydrolase Kau2. The enzyme, which is a member of the alpha/beta-hydrolase fold family, preferentially reacts with the (S,S)-enantiomer of trans-stilbene oxide (TSO) with an E value of similar to 200. The enzyme follows a classical two-step catalytic mechanism with formation of an alkyl-enzyme intermediate in the first step and hydrolysis of this intermediate in a rate-limiting second step. Tryptophan fluorescence quenching during TSO conversion appears to correlate with alkylation of the enzyme. The steady-state data are consistent with (S,S) and (R,R)-TSO being two competing substrates with marked differences in k(cat) and K-M values. The high enantiopreference of the epoxide hydrolase is best explained by pronounced differences in the second-order alkylation rate constant (k(2)/K-S) and the alkyl-enzyme hydrolysis rate k(3) between the (S,S) and (R,R)-enantiomers of TSO. Our data suggest that during conversion of (S,S)-TSO the two active site tyrosines, Tyr(157) and Tyr(259), serve mainly as electrophilic catalysts in the alkylation half-reaction, polarizing the oxirane oxygen of the bound epoxide through hydrogen bond formation, however, without fully donating their hydrogens to the forming alkyl-enzyme intermediate. (c) 2015 Elsevier Inc. All rights reserved.

Název v anglickém jazyce

Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics

Popis výsledku anglicky

A detailed kinetic study based on steady-state and pre-steady-state measurements is described for the highly enantioselective epoxide hydrolase Kau2. The enzyme, which is a member of the alpha/beta-hydrolase fold family, preferentially reacts with the (S,S)-enantiomer of trans-stilbene oxide (TSO) with an E value of similar to 200. The enzyme follows a classical two-step catalytic mechanism with formation of an alkyl-enzyme intermediate in the first step and hydrolysis of this intermediate in a rate-limiting second step. Tryptophan fluorescence quenching during TSO conversion appears to correlate with alkylation of the enzyme. The steady-state data are consistent with (S,S) and (R,R)-TSO being two competing substrates with marked differences in k(cat) and K-M values. The high enantiopreference of the epoxide hydrolase is best explained by pronounced differences in the second-order alkylation rate constant (k(2)/K-S) and the alkyl-enzyme hydrolysis rate k(3) between the (S,S) and (R,R)-enantiomers of TSO. Our data suggest that during conversion of (S,S)-TSO the two active site tyrosines, Tyr(157) and Tyr(259), serve mainly as electrophilic catalysts in the alkylation half-reaction, polarizing the oxirane oxygen of the bound epoxide through hydrogen bond formation, however, without fully donating their hydrogens to the forming alkyl-enzyme intermediate. (c) 2015 Elsevier Inc. All rights reserved.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

CE - Biochemie

OECD FORD obor

—

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2016

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Archives of Biochemistry and Biophysics

ISSN

0003-9861

e-ISSN

—

Svazek periodika

591

Číslo periodika v rámci svazku

FEB 2016

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

10

Strana od-do

66-75

Kód UT WoS článku

000370394400008

EID výsledku v databázi Scopus

2-s2.0-84952361266