Role of Q52 in catalysis of decarboxylation and transamination in dialkylglycine decarboxylase

Emily J. Fogle, Wenshe Liu, See Tarn Woon, John W. Keller, Michael D. Toney

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

Dialkylglycine decarboxylase (DGD) is a pyridoxal phosphate dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. DGD uses stereoelectronic effects to control its unusual reaction specificity. X-ray crystallographic structures of DGD suggest that Q52 is important in maintaining the substrate carboxylate in a stereoelectronically activated position. Here, the X-ray structures of the Q52A mutant and the wild type (WT) DGD-PMP enzymes are presented, as is the analysis of steady-state and half-reaction kinetics of three Q52 mutants (Q52A, Q52I, and Q52E). As expected if stereoelectronic effects are important to catalysis, the steady-state rate of decarboxylation for all three mutants has decreased significantly compared to that of WT. Q52A exhibits an ∼85-fold decrease in kcat relative to that of WT. The rate of the decarboxylation half-reaction decreases ∼105-fold in Q52I and ∼104-fold in Q52E compared to that of WT. Transamination half-reaction kinetics show that Q52A and Q52I have greatly reduced rates compared to that of WT and are seriously impaired in pyridoxamine phosphate (PMP) binding, with KPMP least 50-100-fold greater than that of WT. The larger effect on the rate of L-alanine transamination than of pyruvate transamination in these mutants suggests that the rate decrease is the result of selective destabilization of the PMP form of the enzyme in these mutants. Q52E exhibits near-WT rates for transamination of both pyruvate and L-alanine. Substrate binding has been greatly weakened in Q52E with apparent dissociation constants at least 100-fold greater than that of WT. The rate of decarboxylation in Q52E allows the energetic contribution of stereoelectronic effects, ΔGstereoelectronic, to be estimated to be -7.3 kcal/mol for DGD.

Original languageEnglish (US)
Pages (from-to)16392-16404
Number of pages13
JournalBiochemistry
Volume44
Issue number50
DOIs
StatePublished - Dec 20 2005

ASJC Scopus subject areas

  • Biochemistry

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