Kinetic and chemical mechanism of α-isopropylmalate synthase from Mycobacterium tuberculosis

Luiz Pedro S. De Carvalho, John S. Blanchard

Research output: Contribution to journalArticle

41 Scopus citations

Abstract

Mycobacterium tuberculosis α-isopropylmalate synthase (MtIPMS) catalyzes the condensation of acetyl-coenzyme A (AcCoA) with α-ketoisovalerate (α-KIV) and the subsequent hydrolysis of α-isopropylmalyl-CoA to generate the products CoA and α- isopropylmalate (α-IPM). This is the first committed step in L-leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and 1H-NMR spectroscopy. The α-keto acid specificity of the enzyme is narrow, and the acyl-CoA specificity is absolute for AcCoA. In the absence of α-KIV, MtIPMS does not enolize the α protons of AcCoA but slowly hydrolyzes acyl-CoA analogues. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random bi-bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pKa values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of α-isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate-limiting, although, with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of α-isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. On the basis of these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed.

Original languageEnglish (US)
Pages (from-to)8988-8999
Number of pages12
JournalBiochemistry
Volume45
Issue number29
DOIs
StatePublished - Jul 25 2006

ASJC Scopus subject areas

  • Biochemistry

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