Substrate trapping experiments with P-enolpyruvate carboxykinase from rat and chickel liver

Equilibrium mixtures of P-enolpyruvate carboxykinase (PEPCK) and Mg[α-32P]GTP were mixed with excess oxalacetate and unlabeled MgGTP to establish the fraction of bound GTP which is commited to product formation. At saturating oxalacetate all enzyme-bound [α-32P]GTP was converted to [α-32P]GDP, thus the rate of catalysis is much greater than the dissociation of the PEPCK x MgGTP complex. The concentration of oxalacetate which caused half of the bound GTP to be trapped was 10-20 μM. Control experiments established that no significant conversion of GTP → GDP occurred in the absence of oxalacetate and no evidence was found for a covalent enzyme-phosphoryl complex. Similar results were obtained with both rat and chicken liver PEPCK's. The presence of Mn2+ (in addition to Mg2+) or the presence of KHCO3 had no effect on the GTP trapping efficiency. These experiments confirm kinetic studies which indicated that catalysis is not the slow step for the reaction catalyzed by PEPCK and establish that MgGTP binds in the proper catalytic orientation in the absence of oxalacetate, Mn2+ or HCO3-