Subunit interaction during catalysis. Implications of concentration dependency of oxygen exchanges accompanying oxidative phosphorylation for alternating site cooperativity.

Substrate and product concentrations are shown to have a pronounced effect on the pattern of the isotopic exchange reactions which accompany succinate-driven oxidative phosphorylation by beef heart submitochondrial particles. Rapid removal of ATP by hexokinase and glucose results in a severe inhibition of medium P, e HOH exchange although extensive intermediate exchange still occurs as shown by the loss of phosphate oxygens to water during ATP synthesis. The extent of intermediate exchange is sharply controlled by the level of ADP or of Pi. When either one is limiting, much greater loss of phosphate oxygens to water occurs during ATP synthesis. These observations are readily accommodated by a model having alternating site cooperativity. In this model P, cannot be released to allow a medium Pi 5 HOH exchange without ATP binding at a second conformationally linked site. Prevention of ATP rebinding by a hexokinase-glucose trap thus stops medium P, ti HOH exchange. Conversely, binding of both ADP and Pi at a second site is required for ATP release. Thus, at low ADP or Pi concentrations extensive reversal of bound ATP formation occurs before the bound ATP is released. Such isotopic probes promise to be a powerful tool in the study of alternating site cooperativity for those enzymes where suitable isotopic exchange reactions exist.