Effects of changes in three catalytic residues on the relative stabilities of some of the intermediates and transition states in the citrate synthase reaction

Biochemistry
L C KurzG R Drysdale

Abstract

This work reports the relative importance of the interactions provided by three catalytic residues to individual steps in the mechanism of citrate synthase. When the side chains of any of the residues (H320, D375, and H274) are mutated, the data indicate that they are involved in the stabilization of one or more of the transition/intermediate states in the multistep citrate synthase reaction. H320 forms a hydrogen bond with the carbonyl of oxaloacetate and the alcohols of the citryl-coenzyme A and citrate products. Enzymes substituted at H320 (Q, G, N, and R) have reaction profiles for which the condensation reaction is cleanly rate determining. None of these mutants can activate the carbonyl of oxaloacetate by polarization. All these mutants catalyze the necessary proton transfer from the methyl group of acetyl-coenzyme A only poorly, a process which occurs in a structurally separate site. Furthermore, all H320 mutants hydrolyze the citryl-coenzyme A intermediate significantly more slowly than does the wild-type. D375 is the base removing the proton of acetyl-coenzyme A. D375E and D375G have greatly diminished ability to catalyze proton transfer from acetyl-CoA. The D375 mutants polarize the oxaloacetate carbonyl as well as wi...Continue Reading

References

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Citations

Apr 11, 2008·Chemical Communications : Chem Comm·Marc W van der KampAdrian J Mulholland
May 20, 2015·Molecular Cell·Kunio NakatsukasaTakumi Kamura
Sep 18, 2004·American Journal of Physiology. Regulatory, Integrative and Comparative Physiology·Anne C DalzielChristopher D Moyes
Oct 24, 2002·The Journal of Biological Chemistry·Clare V SmithKerstin Höner zu Bentrup

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