Chemical Mechanism of the Branched-Chain Aminotransferase IlvE from Mycobacterium tuberculosis

Tathyana M. Amorim Franco, Subray Hegde, John S. Blanchard

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The biosynthetic pathway of the branched-chain amino acids is essential for Mycobacterium tuberculosis growth and survival. We report here the kinetic and chemical mechanism of the pyridoxal 5′-phosphate (PLP)-dependent branched-chain aminotransferase, IlvE, from M. tuberculosis (MtIlvE). This enzyme is responsible for the final step of the synthesis of the branched-chain amino acids isoleucine, leucine, and valine. As seen in other aminotransferases, MtIlvE displays a ping-pong kinetic mechanism. pK values were identified from the pH dependence on V as well as V/K, indicating that the phosphate ester of the PLP cofactor, and the α-amino group from l-glutamate and the active site Lys204, play roles in acid-base catalysis and binding, respectively. An intrinsic primary kinetic isotope effect was identified for the α-C-H bond cleavage of l-glutamate. Large solvent kinetic isotope effect values for the ping and pong half-reactions were also identified. The absence of a quininoid intermediate in combination with the Dkobs in our multiple kinetic isotope effects under single-turnover conditions suggests a concerted type of mechanism. The deprotonation of C2 of l-glutamate and the protonation of C4′ of the PLP cofactor happen synchronously in the ping half-reaction. A chemical mechanism is proposed on the basis of the results obtained here.

Original languageEnglish (US)
Pages (from-to)6295-6303
Number of pages9
JournalBiochemistry
Volume55
Issue number45
DOIs
StatePublished - Nov 15 2016

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Mycobacterium tuberculosis
Pyridoxal Phosphate
Kinetics
Isotopes
Glutamic Acid
Branched Chain Amino Acids
Deprotonation
Isoleucine
Protonation
Biosynthetic Pathways
Valine
Transaminases
Catalysis
Leucine
Catalytic Domain
Esters
Phosphates
branched-chain-amino-acid transaminase
Acids
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chemical Mechanism of the Branched-Chain Aminotransferase IlvE from Mycobacterium tuberculosis. / Amorim Franco, Tathyana M.; Hegde, Subray; Blanchard, John S.

In: Biochemistry, Vol. 55, No. 45, 15.11.2016, p. 6295-6303.

Research output: Contribution to journalArticle

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