Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC).
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-alpha-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 approximately 306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of approximately 9.4 and approximately 5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.
Fan
Fan
F
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Luxenburger
Andreas
A
Painter
Gavin F
GF
Blanchard
John S
JS
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D005944:Glucosamine / Q000737:chemistry / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D015394:Molecular Structure; D020102:Mycobacterium smegmatis / Q000201:enzymology*
D011756:Diphosphates; D006020:Glycopeptides; C089265:mycothiol; D007294:Inositol; D000255:Adenosine Triphosphate; D008025:Ligases; D003545:Cysteine; D005944:Glucosamine