Stereochemical course of thiophosphoryl transfer catalyzed by cytosolic phosphoenolpyruvate carboxykinase

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Abstract
Rat liver cytosolic phosphoenolypyruvate 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 .mu.mol min-1 (mg of protein)-1, respectively, compared with the corresponding values of 0.168 mM and 76 .mu.mol 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.