Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase.
Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast and plants. Pantothenate synthetase from Mycobacterium tuberculosis was expressed in E. coli, purified to homogeneity, and found to be a homodimer with a subunit molecular mass of 33 kDa. Initial velocity, product, and dead-end inhibition studies showed the kinetic mechanism of pantothenate synthetase to be Bi Uni Uni Bi Ping Pong, with ATP binding followed by D-pantoate binding, release of PP(i), binding of beta-alanine, followed by the release of pantothenate and AMP. Michaelis constants were 0.13, 0.8, and 2.6 mM for D-pantoate, beta-alanine, and ATP, respectively, and the turnover number, k(cat), was 3.4 s(-1). The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by (31)P NMR spectroscopy of [(18)O]AMP produced from (18)O transfer using [carboxyl-(18)O]pantoate. Single-turnover reactions for the formation of pyrophosphate and pantothenate were determined using rapid quench techniques, and indicated that the two half-reactions occurred with maximum rates of 1.3 +/- 0.3 and 2.6 +/- 0.3 s(-)(1), respectively, consistent with pantoyl adenylate being a kinetically competent intermediate in the pantothenate synthetase reaction. These data also suggest that both half-reactions are partially rate-limiting. Reverse isotope exchange of [(14)C]-beta-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products.
Zheng
R
R
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Blanchard
J S
JS
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D006885:Hydroxybutyrates; D007477:Ions; D011994:Recombinant Proteins; C016031:pantoic acid; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C022318:pantothenate synthetase; D000409:Alanine; D010100:Oxygen