Evaluation of the partitioning of bound inorganic phosphate during medium and intermediate phosphate in equilibrium water oxygen exchange reactions of yeast inorganic pyrophosphatase.

Abstract

During the rapid exchange of oxygens of Pi with water catalyzed by yeast inorganic pyrophosphatase [EC 3.6.1.1], Pi at the catalytic site may either dissociate or undergo reversible loss of an oxygen to water. The effective partitioning of bound Pi during exchange starting with medium Pi containing 18O in all 4 oxygens was evaluated by mass spectral analysis of the change in the distribution of Pi species containing 0-4 18O oxygens per Pi. This analysis indicates that the rate of Pi release from the enzyme is only 1.4 times faster than the rate of reformation of the anhydrous intermediate. A similar partitioning of bound Pi is observed during PPi hydrolysis, indicating that hydrolysis and medium exchange have common intermediates. The approach should be applicable to the study of related phosphate oxygen exchanges.