The hypothesis that Escherichia coli succinyl-CoA synthetase functions by a cooperative alternating sites mechanism is based largely on the results of [18O]phosphate exchange experiments (Bild, G.S., Janson, C.A., and Boyer, P.D. (1980) J. Biol. Chem. 255, 8109-8115). In those experiments, [18O]P(i) ⇆ succinate (predominantly) exchange appeared to proceed at greater rates (relative to the apparent amount of succinyl-CoA released from the enzyme) at low ATP in incubations containing ATP, CoA, succinate, [18O]P(i), 0.48 M hydroxylamine (as a succinyl-CoA trap), and a pyruvate kinase-lactase dehydrogenase ADP trap. The conclusion arrived at was that succinyl-CoA binding at one site was inversely related to ATP binding at the second site. Thus, the residence time of succinyl-CoA binding at a site would be longer at lower ATP concentrations. Our experiments show that, under the incubation conditions described by Bild et al. (Bild, G.S., Janson, C.A., and Boyer, P.D. (1980) J. Biol. Chem. 255, 8109-8115), succinyl-CoA is not efficiently trapped. Thus, at ATP concentrations from 3.6 to 150 μM, concentrations of succinyl-CoA from 13 to 78 μM were observed. Succinate ⇆ succinyl-CoA exchange reactions carried out in this range of ATP and subsaturating succinyl-CoA concentrations were found to be markedly inhibited by the addition of the ADP trap. This inhibition was more pronounced at higher ATP levels. At a saturating succinyl-CoA concentration (1.5 mM), addition of the ADP trap actually stimulated succinate ⇆ succinyl-CoA exchange. Under these conditions, ATP ⇆ P(i) exchange was greatly depressed. These results are interpreted as follows. ADP is required for optimal binding of succinyl-CoA, but only when the latter is present at subsaturating concentrations; thus, the ADP trap inhibits the reaction. ATP exerts its stimulatory action on succinate ⇆ succinyl-CoA exchange through an 'other site' effect, i.e. in binding to the noncatalytic site of succinyl-CoA synthetase, it facilitates binding and release of succinyl-CoA at the catalytic site. ATP may also exert negative effects by inhibiting other site binding of ATP or by interfering with same site succinyl-CoA binding at subsaturating concentrations of the latter. These data support the notion that a half-sites mechanism applies to succinyl-CoA synthetase, but suggest that the [18O]P(i) ⇆ succinate exchange data which have been instrumental in development of the cooperative alternating sites hypothesis should be re-evaluated.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology