Stereochemical course of thiophosphoryl transfer catalyzed by cytosolic phosphoenolpyruvate carboxykinase.
Rat liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) utilizes inosine 5'-(3-thiotriphosphate) (ITP gamma S) as an excellent substrate, with Km and V values of 0.08 mM and 37 mumol min-1 (mg of protein)-1, respectively, compared with the corresponding values of 0.168 mM and 76 mumol min-1 (mg of protein)-1 for ITP. Thus, the V/Km values for the two substrates are the same. Reaction of (RP)-[gamma-18O2]ITP gamma S with oxalacetate catalyzed by cytosolic PEPCK produces (SP)-thio[18O]phosphoenolpyruvate. Therefore, thiophosphoryl transfer catalyzed by this enzyme proceeds with overall inversion of configuration at P. The reaction mechanism involves an uneven number of phosphotransfer steps, most likely a single step transfer between bound substrates. The results do not support the involvement of a phosphoryl enzyme intermediate in the mechanism.
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D007288:Inosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010103:Oxygen Isotopes; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D010103:Oxygen Isotopes; D007293:Inosine Triphosphate; D007288:Inosine; C050291:inosine 5'-(3-thiotriphosphate); D010729:Phosphoenolpyruvate Carboxykinase (GTP)