Stereoselectivity of interaction of phosphoenolpyruvate analogs with various phosphoenolpyruvate-utilizing enzymes

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Abstract
The halogenated phosphoenolpyruvate analogs (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogs were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolypyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase [PEP-CK chicken liver], enolase [yeast] and pyruvate phosphate dikinase [Bacteroides symbiosus] was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrated activity with the enzyme pyruvate kinase [rabbit muscle], however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogs with the enzymes PEP-CK enolase and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured K1 values and analog binding resembles phosphoenolpyruvate binding. With PEP-CK, the KI .noteq. K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analog decomposing much faster than the fluoro analogs.
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