Solvent Isotope Effect on the Reaction Catalysed by the Pyruvate Dehydrogenase Complex from Escherichia coli

Solvent Isotope Effect on the Reaction Catalysed by the Pyruvate Dehydrogenase Complex from... Abstract The pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide. The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction. The pH profile and, accordingly, the δpK1 and δpK2 values were shifted by 0.6 units to higher pL values. A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0%. This is an indication for a single proton transfer. It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1 nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue. The proton of its carboxylic group exchanges very fast with deuterons of the solvent. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biological Chemistry de Gruyter

Solvent Isotope Effect on the Reaction Catalysed by the Pyruvate Dehydrogenase Complex from Escherichia coli

Biological Chemistry, Volume 384 (4) – Apr 10, 2003

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Publisher
de Gruyter
Copyright
Copyright © 2003 by the
ISSN
1431-6730
DOI
10.1515/BC.2003.075
pmid
12751797
Publisher site
See Article on Publisher Site

Abstract

Abstract The pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide. The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction. The pH profile and, accordingly, the δpK1 and δpK2 values were shifted by 0.6 units to higher pL values. A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0%. This is an indication for a single proton transfer. It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1 nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue. The proton of its carboxylic group exchanges very fast with deuterons of the solvent.

Journal

Biological Chemistryde Gruyter

Published: Apr 10, 2003

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