Positional isotope exchange analysis of the pantothenate synthetase reaction.
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 the addition of pyrophosphatase; 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, beta-alanine, was increased. This observation is consistent with a kinetic mechanism that requires the binding of beta-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.
Williams
LaKenya
L
Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, Texas 77842-3012, USA.
Zheng
Renjian
R
Blanchard
John S
JS
Raushel
Frank M
FM
D000255:Adenosine Triphosphate / Q000378:metabolism; D007553:Isotope Labeling; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010103:Oxygen Isotopes; D010453:Peptide Synthases / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D017680:Potassium Compounds / Q000378:metabolism
D010103:Oxygen Isotopes; D010710:Phosphates; D017680:Potassium Compounds; D000255:Adenosine Triphosphate; C013216:potassium phosphate; D010453:Peptide Synthases; C022318:pantothenate synthetase