SUBUNIT INTERACTION DURING CATALYSIS - ATP MODULATION OF CATALYTIC STEPS IN THE SUCCINYL-COA SYNTHETASE REACTION

Abstract

A new approach for assessing if catalytic cooperativity may occur between subunits was applied to succinyl-CoA synthetase. This is based on the extent of O2 exchange between medium [18O]Pi and succinate per molecule of ATP cleaved during steady state succinyl-CoA synthesis. Suitable traps are used to remove succinyl-CoA and ADP as soon as they are released to the medium. With the Escherichia coli enzyme, which has an .alpha.2.beta.2 structure, a pronounced increase in O2 exchange per ATP cleaved occurs as ATP concentration is lowered. In contrast, when the CoA concentration is varied, the O2 exchange per molecule of product formed remains constant. Also, with the pig heart enzyme, which is shown to retain its .alpha..beta. structure during catalysis and thus has only 1 catalytic site, no modulation of O2 exchange by ATP concentration is observed. The binding of an ATP either promotes the dissociation of bound succinyl-CoA or decreases its participation in exchange. Measurement of the distribution of [18O]Pi species found as exchange occurs shows that only 1 catalytic sequence is involved in exchange at various ATP concentrations. These observations along with other controls and results eliminate most other explanations of the ATP modulation of the exchange and suggest that binding of ATP at 1 catalytic site promotes catalytic events at an alternate catalytic site.