Pre-steady-state kinetic analysis of the reactions of alternate substrates with dialkylglycine decarboxylase

Shaoxian Sun, Carey K. Bagdassarian, Michael D. Toney

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

The pre-steady-state kinetics of the half-reactions of several substrates with dialkylglycine decarboxylase are examined by multiwavelength kinetics and global analysis. The substrates examined fall into two groups: those that exhibit simple, monophasic kinetics and those that exhibit biphasic kinetics. The rate of the AIB half-reaction is likely limited by the decarboxylation step based on the simple kinetics and spectra obtained from global analysis. The spectra for the first species in the transamination half-reactions of L-alanine and L-aminobutyrate show long-wavelength absorption characteristic of a carbanionic quinonoid intermediate. This demonstrates that formation of the external aldimine intermediates and abstraction of the C(α) protons from them are rapid. The reactions of the slower substrates L-phenylglycine and 1-aminocyclohexane-1-carboxylate may have external aldimine formation as the rate-determining step. The biphasic reactions of 2-methyl-2-aminomalonate, 1-aminocyclopentane-1-carboxylate, isopropylamine, and glycine all have external aldimine formation as the rapid observable step, based on the spectral changes observed in absorption and circular dichroism measurements. 2-Methyl-2-aminomalonate reacts ~104-fold slower than does AIB with dialkylglycine decarboxylase, compared to ~105- fold faster with coenzyme in solution. It is proposed that this radical reactivity reversal is due to a slow protein conformational change that is a prerequisite to decarboxylation of MAM, which occurs rapidly thereafter. Circular dichroism measurements on active site bound coenzyme provide evidence supporting this proposal. The binding of the noncovalent inhibitors pyruvate or lactate or the covalently binding inhibitor 1-aminocyclopropane- 1-carboxylate all induce a slow change in coenzyme circular dichroism that quantitatively parallels the slow decarboxylation of 2-methyl-2- aminomalonate. Fast circular dichroism changes are seen in the mixing time of these measurements for both 1-aminocyclopropane-1-carboxylate and 2-methyl- 2-aminomalonate, indicating rapid external aldimine formation on this longer time scale.

Original languageEnglish (US)
Pages (from-to)3876-3885
Number of pages10
JournalBiochemistry
Volume37
Issue number11
DOIs
StatePublished - Mar 17 1998
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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