Mechanism of the Reaction Catalyzed by Mandelate Racemase. 1. Chemical and Kinetic Evidence for a Two-Base Mechanism

The fate of the α-hydrogen of mandelate in the reaction catalyzed by mandelate racemase has been investigated by a mass spectroscopic method. The method entails the incubation of (R)- or (S)-[α-¹H] mandelate in buffered D₂O to a low extent of turnover (about 5–8%), esterification of the resulting mixture of mandelates with diazomethane, derivatization of the methyl esters with a chiral derivatizing agent, and quantitation of the isotope content of the α-hydrogen of both substrate and product by gas chromatography/mass spectrometric analysis. No significant substrate-derived α-protium was found in the product for racemization in either direction. In addition, in the (R) to (S) direction almost no exchange (≤0.4%) of the α-hydrogen in the remaining (R) substrate pool occurred, but in the (S) to (R) direction 3.5–5.1% exchange of the α-hydrogen in the remaining substrate (after 5.1–7.2% net turnover) was found. Qualitatively similar results were obtained in the (S) to (R) direction in H₂O when (S)-[α-²H]mandelate was used as substrate. In other experiments, an overshoot in the progress curve was observed when the racemization of either enantiomer of [α-¹H] mandelate in D₂O was monitored by following the change in ellipticity of the reaction mixture; the magnitude of the overshoot was greater in the (R) to (S) than in the (S) to (R) direction. All of the available data indicate that the reaction catalyzed by mandelate racemase proceeds by a two-base mechanism, in contrast to earlier proposals.