Mandelate racemase from Pseudomonas putida. Absence of detectable intermolecular proton transfer accompanying racemization

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Abstract
An equimolar mixture of DL-[.alpha.-2H]- and DL-[.alpha.-13C]mandelate, when incubated with mandelate racemase (EC 5.1.2.2), shows conversion of singly labeled mandelate to unlabeled mandelate due to solvent exchange of the .alpha. proton, while the level of doubly labeled mandelate remains at a constant low level. Similarly, an equimolar mixture of unlabeled and DL-[.alpha.-2H,.alpha.-13C]mandelate, when incubated with the enzyme, shows conversion of doubly labeled mandelate to singly labeled mandelate, due to solvent exchange, while the level of unlabeled mandelate remains constant at 50%. Incubation of an equimolar mixture of DL-[.alpha.-3H]mandelate and DL-p-chloromandelate, both with similar properties as substrates for mandelate racemase, showed solvent exchange of the .alpha.-3H of mandelate, but no 3H appeared in the p-chloromandelate. Mandelate racemase apparently does not catalyze an intermolecular proton transfer to achieve racemization. These data are necessary, but not sufficient, results to indicate that mandelate racemase operates via a 1-acceptor mechanism in which the proton abstracted from 1 stereochemical face of a substrate molecule is returned to the opposite face of the same C of the substrate molecule.