Effect of pH on Hydride Transfer by Escherichia coli Dihydrofolate Reductase

Loveridge, Edric Joel and Allemann, Rudolf Konrad ORCID: https://orcid.org/0000-0002-1323-8830 2011. Effect of pH on Hydride Transfer by Escherichia coli Dihydrofolate Reductase. ChemBioChem 12 (8) , pp. 1258-1262. 10.1002/cbic.201000794

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Abstract

The kinetic isotope effect (KIE) on hydride transfer in the reaction catalysed by dihydrofolate reductase from Escherichia coli (EcDHFR) is known to be temperature dependent at pH 7, but essentially independent of temperature at elevated pH. Here, we show that the transition from the temperature-dependent regime to the temperature-independent regime occurs sharply between pH 7.5 and 8. The activation energy for hydride transfer is independent of pH. The mechanism leading to the change in behaviour of the KIEs is not clear, but probably involves a conformational change in the enzyme brought about by deprotonation of a key residue (or residues) at high pH. The KIE on hydride transfer at low pH suggests that the rate constant for the reaction is not limited by a conformational change to the enzyme under these conditions. The effect of pH on the temperature dependence of the rate constants and KIEs for hydride transfer catalysed by EcDHFR suggests that enzyme motions and conformational changes do not directly influence the chemistry, but that the reaction conditions affect the conformational ensemble of the enzyme prior to reaction and control the reaction though this route.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Cardiff Catalysis Institute (CCI) Chemistry
Subjects:	Q Science > QD Chemistry
Uncontrolled Keywords:	dihydrofolate reductase; enzyme catalysis; hydrides; isotope effects; kinetics; pH
Publisher:	Wiley-Blackwell
ISSN:	1439-4227
Funders:	BBSRC
Last Modified:	18 Oct 2022 13:35
URI:	https://orca.cardiff.ac.uk/id/eprint/14505

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