Rapid kinetic and isotopic studies on dialkylglycine decarboxylase

X. Zhou, X. Jin, R. Medhekar, X. Chen, T. Dieckmann, M. D. Toney

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Abstract

The two half-reactions of the pyridoxal 5′-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761-5769]. The half-reaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steady-state kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half-reaction with the potassium form of DGD-PLP is 25 s-1, while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s-1. The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s-1. The spectral data indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed in the dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-13C]AIB kinetic isotope effect (KIE) on kcat for the steady-state reaction is 1.043±0.003, while a value of 1.042±0.009 was measured for the AIB half-reaction. The secondary KIE measured for the AIB decarboxylation half-reaction with [C4′-2H]PLP is 0.92±0.02. The primary [2-2H]-L-alanine KIE on the transamination half-reaction is unity. Small but significant solvent KIEs are observed on kcat and kcat/KM for both substrates, and the proton inventories are linear in each case. NMR measurements of C2-H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted Cα-decarboxylation/C4′-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half-reactions are constructed.

Original languageEnglish (US)
Pages (from-to)1367-1377
Number of pages11
JournalBiochemistry
Volume40
Issue number5
DOIs
StatePublished - Feb 6 2001

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2,2-dialkylglycine decarboxylase
Pyridoxal Phosphate
Decarboxylation
Alanine
Kinetics
Isotopes
Protonation
Pyruvic Acid
Kinetic parameters
Rate constants
Potassium
Alkali Metals
Biochemistry
Substrates
Reaction kinetics
Metal ions
Conformations
Protons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Zhou, X., Jin, X., Medhekar, R., Chen, X., Dieckmann, T., & Toney, M. D. (2001). Rapid kinetic and isotopic studies on dialkylglycine decarboxylase. Biochemistry, 40(5), 1367-1377. https://doi.org/10.1021/bi001237a

Rapid kinetic and isotopic studies on dialkylglycine decarboxylase. / Zhou, X.; Jin, X.; Medhekar, R.; Chen, X.; Dieckmann, T.; Toney, M. D.

In: Biochemistry, Vol. 40, No. 5, 06.02.2001, p. 1367-1377.

Research output: Contribution to journalArticle

Zhou, X, Jin, X, Medhekar, R, Chen, X, Dieckmann, T & Toney, MD 2001, 'Rapid kinetic and isotopic studies on dialkylglycine decarboxylase', Biochemistry, vol. 40, no. 5, pp. 1367-1377. https://doi.org/10.1021/bi001237a
Zhou X, Jin X, Medhekar R, Chen X, Dieckmann T, Toney MD. Rapid kinetic and isotopic studies on dialkylglycine decarboxylase. Biochemistry. 2001 Feb 6;40(5):1367-1377. https://doi.org/10.1021/bi001237a
Zhou, X. ; Jin, X. ; Medhekar, R. ; Chen, X. ; Dieckmann, T. ; Toney, M. D. / Rapid kinetic and isotopic studies on dialkylglycine decarboxylase. In: Biochemistry. 2001 ; Vol. 40, No. 5. pp. 1367-1377.
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