Analysis of ping-pong reaction mechanisms by positional isotope exchange. Application to galactose-1-phosphate uridyltransferase
A new positional isotope exchange method has been developed that can be used for the analysis of enzyme-catalyzed reactions which have ping-pong kinetic mechanisms. The technique can be used to measure the relative rates of ligand dissociation from enzyme-product complexes. Enzyme is incubated with the labeled substrate and an excess of the corresponding unlabeled product. The partitioning of the enzyme-product complex back toward free enzyme is determined from the rate of positional isotope exchange within the original labeled substrate. The partitioning of the enzyme-product complex forward toward free enzyme is determined from the rate of formation of totally unlabeled substrate. It has been shown that the ratio of the two rates provides a lower limit for the release of product from the enzyme-product complex. The technique has been applied to the reaction catalyzed by galactose-1-phosphate uridyltransferase. The lower limit for the release of glucose 1-phosphate from the uridyl-enzyme relative to the maximal velocity of the reverse reaction was determined to be 3.4 +/- 0.5.
- Research Organization:
- Texas AandM Univ., College Station
- OSTI ID:
- 6075849
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 262:25
- Country of Publication:
- United States
- Language:
- English
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