Amino Acids Allosterically Regulate the Thiamine Diphosphate-dependent α-Keto Acid Decarboxylase from Mycobacterium tuberculosis

Ralph Golbik,Anja Schütz,Michael Spinka,Stephan König,Kai Tittmann,Tobias Werther,Gerhard Hübner,Carmen Mrestani-Klaus

doi:10.1074/jbc.m706569200

Ralph Golbik, Anja Schütz + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m706569200

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Abstract

The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

Highlights

Based on their substrate specificity, non-oxidative ThDP-dependent ␣-keto acid decarboxylases yielding aldehydes can be subdivided into various groups
Indolepyruvate decarboxylases (IPDCs) and phenylpyruvate decarboxylases are key enzymes in the biosynthesis of the plant hormones indoleacetic acid and phenylacetic acid, which are derived from the aromatic amino acids tryptophan and phenylalanine, respectively [5, 6]
In this study we provide kinetic evidence that a ThDP-dependent ␣-keto acid decarboxylase, which participates in amino acid degradation via the Ehrlich pathway, is subject to allosteric activation

Summary

Introduction

Based on their substrate specificity, non-oxidative ThDP-dependent ␣-keto acid decarboxylases yielding aldehydes can be subdivided into various groups. 1H NMR Experiments—To investigate the influence of activators on the deprotonation rate constant at the C2 atom of enzyme-bound ThDP, H/D exchange kinetics were monitored in presence and absence of an amino acid at 10 mg of MtKDC/ ml, 30 °C, and pH 6.5 [41]. Km f ax is the rate constant for the activation in forward direction at a given substrate concentration under amino acid saturation.

Results

Conclusion