Positional isotope exchange analysis of the pantothenate synthetase reaction

LaKenya Williams, Renjian Zheng, John S. Blanchard, Frank M. Raushel

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Pantothenate synthetase from Mycobacterium tuberculosis catalyzes the formation of pantothenate from ATP, D-pantoate, and β-alanine. The formation of a kinetically competent pantoyl-adenylate intermediate was established by the observation of a positional isotope exchange (PIX) reaction within 18O-labeled ATP in the presence of D-pantoate. When [βγ-18O6]-ATP was incubated with pantothenate synthetase in the presence of D-pantoate, an 18O label gradually appeared in the αβ-bridge position from both the β- and the γ-nonbridge positions. The rates of these two PIX reactions were followed by 31P NMR spectroscopy and found to be identical. These results are consistent with the formation of enzymebound pantoyl-adenylate and pyrophosphate upon the mixing of ATP, D-pantoate, and enzyme. In addition, these results require the complete torsional scrambling of the two phosphoryl groups of the labeled pyrophosphate product. The rate of the PIX reaction increased as the D-pantoate concentration was elevated and then decreased to zero at saturating levels of D-pantoate. These inhibition results support the ordered binding of ATP and D-pantoate to the enzyme active site. The PIX reaction was abolished with the addition of pyrophosphatase; thus, PPi must be free to dissociate from the active site upon formation of the pantoyladenylate intermediate. The PIX reaction rate diminished when the concentrations of ATP and D-pantoate were held constant and the concentration of the third substrate, β-alanine, was increased. This observation is consistent with a kinetic mechanism that requires the binding of β-alanine after the release of pyrophosphate from the active site of pantothenate synthetase. Positional isotope exchange reactions have therefore demonstrated that pantothenate synthetase catalyzes the formation of a pantoyl-adenylate intermediate upon the ordered addition of ATP and pantoate.

Original languageEnglish (US)
Pages (from-to)5108-5113
Number of pages6
JournalBiochemistry
Volume42
Issue number17
DOIs
StatePublished - May 6 2003

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pantothenate synthetase
Isotopes
Adenosine Triphosphate
Alanine
Catalytic Domain
4 alpha-glucanotransferase
Pyrophosphatases
Enzymes
Mycobacterium tuberculosis
Nuclear magnetic resonance spectroscopy
Reaction rates
Labels
Magnetic Resonance Spectroscopy

ASJC Scopus subject areas

  • Biochemistry

Cite this

Positional isotope exchange analysis of the pantothenate synthetase reaction. / Williams, LaKenya; Zheng, Renjian; Blanchard, John S.; Raushel, Frank M.

In: Biochemistry, Vol. 42, No. 17, 06.05.2003, p. 5108-5113.

Research output: Contribution to journalArticle

Williams, LaKenya ; Zheng, Renjian ; Blanchard, John S. ; Raushel, Frank M. / Positional isotope exchange analysis of the pantothenate synthetase reaction. In: Biochemistry. 2003 ; Vol. 42, No. 17. pp. 5108-5113.
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