Multiple hydrogen kinetic isotope effects for enzymes catalyzing exchange with solvent: Application to alanine racemase

M. Ashley Spies, Michael D. Toney

Research output: Contribution to journalArticlepeer-review

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Alanine racemase catalyzes the pyridoxal phosphate-dependent interconversion of the D- and L-isomers of alanine. Previous studies have shown that the enzyme employs a two-base mechanism in which Lys39 and Tyr265 are the acid/base catalysts. It is thus possible that stereoisomerization of the external aldimine intermediates occurs through a concerted double proton transfer without the existence of a distinct carbanionic intermediate. This possibility was tested by the application of multiple kinetic isotope effect (KIE) methodology to alanine racemase. The mutual dependence of primary substrate and solvent deuterium KIEs has been measured using equilibrium perturbation-type experiments. The conceptually straightforward measurement of the substrate KIE in H2O is complemented with a less intuitive protium washout perturbation-type measurement in D2O. The primary substrate KIE in the D → L direction at 25 °C is reduced from 1.297 in H2O to 1.176 in D2O, while in the L → D direction it is reduced from 1.877 in H2O to 1.824 in D2O. Similar reductions are also observed at 65 °C, the temperature to which the Bacillus stearothermophilus enzyme is adapted. These data strongly support a stepwise racemization of stereoisomeric aldimine intermediates in which a substrate-based carbanion is an obligatory intermediate. The ionizations observed in kcat/KM pH profiles have been definitively assigned based on the ΔHion values of the observed pKa's with alanine and on the pH dependence of kcat/KM for the alternative substrate serine. The acidic pKa in the bell-shaped curve is due to the phenolic hydroxyl of Tyr265, which must be unprotonated for reaction with either isomer of alanine. The basic pKa is due to the substrate amino group, which must be protonated to react with Tyr265-unprotonated enzyme. A detailed reaction mechanism incorporating these results is proposed.

Original languageEnglish (US)
Pages (from-to)5099-5107
Number of pages9
Issue number17
StatePublished - May 6 2003

ASJC Scopus subject areas

  • Biochemistry


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