Abstract
Pantothenate synthetase from Mycobacterium tuberculosis catalyzes the formation of pantothenate from ATP, D-pantoate, and -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 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 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 the addition of inorganic pyrophosphatase and thus PP[i] must be free to dissociate from the active site upon formation of the pantoyl-adenylate 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 D-pantoate. The third substrate, β-alanine, binds to the enzyme upon the release of pyrophosphate from the active site.
Williams, Lakenya Flatreese (2003). The analysis of the pantothenate synthetase reaction by positional isotope exchange. Master's thesis, Texas A&M University. Available electronically from
https : / /hdl .handle .net /1969 .1 /ETD -TAMU -2003 -THESIS -W366.