Positional isotope exchange analysis of the pantothenate synthetase reaction

Biochemistry
LaKenya WilliamsFrank M Raushel

Abstract

Pantothenate synthetase from Mycobacterium tuberculosis catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine. The formation of a kinetically competent pantoyl-adenylate intermediate was established by the observation of a positional isotope exchange (PIX) reaction within (18)O-labeled ATP in the presence of d-pantoate. When [betagamma-(18)O(6)]-ATP was incubated with pantothenate synthetase in the presence of d-pantoate, an (18)O label gradually appeared in the alphabeta-bridge position from both the beta- and the gamma-nonbridge positions. The rates of these two PIX reactions were followed by (31)P NMR spectroscopy and found to be identical. These results are consistent with the formation of enzyme-bound 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 t...Continue Reading

Related Concepts

KD2PO4
Pantoate-beta-alanine ligase
Adenosine Triphosphate, Chromium Ammonium Salt
Isotope Labeling, Stable
Mycobacterium tuberculosis H37Rv
In Vivo NMR Spectroscopy
Oxygen Isotopes
Peptide synthase
Orthophosphate
Potassium Compounds

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