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

R. Zheng, J. S. Blanchard

Research output: Contribution to journalArticlepeer-review

84 Scopus citations

Abstract

Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and β-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 PPi, binding of β-alanine, followed by the release of pantothenate and AMP. Michaelis constants were 0.13, 0.8, and 2.6 mM for D-pantoate, β-alanine, and ATP, respectively, and the turnover number, kcat, was 3.4 s-1. The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by 31P NMR spectroscopy of [18O]AMP produced from 18O transfer using [carboxyl-18O]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 [14C]-β-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products.

Original languageEnglish (US)
Pages (from-to)12904-12912
Number of pages9
JournalBiochemistry
Volume40
Issue number43
DOIs
StatePublished - Oct 31 2001

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase'. Together they form a unique fingerprint.

Cite this