Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC)

Fan Fan, Andreas Luxenburger, Gavin F. Painter, John S. Blanchard

Research output: Contribution to journalArticle

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Abstract

Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido- 2-deoxy-α-D-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-MshD. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady-state and pre-steady-state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady-state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s -1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5′-O-[N-(L-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of ∼306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of ∼9.4 and ∼5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.

Original languageEnglish (US)
Pages (from-to)11421-11429
Number of pages9
JournalBiochemistry
Volume46
Issue number40
DOIs
Publication statusPublished - Oct 9 2007

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ASJC Scopus subject areas

  • Biochemistry

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