Control of Phosphoenolpyruvate Synthesis by Substrate Level Phosphorylation in Guinea Pig Liver Mitochondria

Abstract

Control of P-enolpyruvate synthesis has been studied in guinea pig liver mitochondria with respect to the relative rates of GTP generation directly from substrate level phosphorylation or indirectly from ATP via nucleoside diphosphate kinase. With malate or pyruvate plus malate as substrates, P-enolpyruvate production was greatly decreased in State 3 by addition of fluorocitrate (an inhibitor of aconitase) to prevent flux through substrate level phosphorylation. On the other hand, when ATP production by the electron transport chain was prevented by use of an uncoupling agent (together with oligomycin to inhibit ATPase activity), rates of P-enolpyruvate production from α-ketoglutarate were the same as in State 3, and were also unaffected by addition of fluorocitrate. In the absence of oxidative phosphorylation and with fluorocitrate present together with malate or pyruvate plus malate, addition of exogenous ATP was much less effective than α-ketoglutarate in promoting P-enolpyruvate synthesis. Intramitochondrial GTP levels and rates of P-enolpyruvate formation were consistently lower in State 4, State 3, and the uncoupled plus oligomycin state with pyruvate plus malate than with α-ketoglutarate plus malate as substrate when fluorocitrate was present, indicating a limitation in the rate of phosphorylation of GDP by intramitochondrial ATP for optimal rates of P-enolpyruvate synthesis. The maximal rate of conversion of ATP to GTP by nucleoside diphosphate kinase in the intact mitochondria was estimated to be 2 to 3 nmoles per min per mg of protein compared with a maximal rate of P-enolpyruvate formation of about 22 nmoles per min per mg of protein obtained with α-ketoglutarate plus malate as substrate. It is concluded that provision of GTP by substrate level phosphorylation is essential to support rates of P-enol-pyruvate formation by guinea pig liver mitochondria greater than 3 nmoles per min per mg of protein.