Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase

Biochemistry. 2001 Oct 30;40(43):12904-12. doi: 10.1021/bi011522+.


Pantothenate synthetase (EC, 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Monophosphate / metabolism
  • Adenosine Triphosphate / metabolism
  • Alanine / pharmacology
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Hydroxybutyrates / chemistry
  • Hydroxybutyrates / metabolism
  • Ions / metabolism
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Chemical
  • Mycobacterium tuberculosis / enzymology*
  • Oxygen / metabolism
  • Peptide Synthases / chemistry*
  • Peptide Synthases / genetics
  • Peptide Synthases / metabolism
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Temperature
  • Time Factors


  • Hydroxybutyrates
  • Ions
  • Recombinant Proteins
  • pantoic acid
  • Adenosine Monophosphate
  • Adenosine Triphosphate
  • Peptide Synthases
  • pantothenate synthetase
  • Alanine
  • Oxygen