Abstract
1H-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) c catalysed hydrogen-deuterium exchange of the alpha-protons of L- and D-alanine and -tryptophan. The first-order and second-order rate constants for exchange have been determined at pH 7.8 in the presence and absence of the allosteric effector, DL-alpha-glycerol 3-phosphate. In the presence of DL-alpha-glycerol 3-phosphate the stereospecificity of the tryptophan synthase-catalyzed first-order exchange rates was in the order tryptophan > alanine > glycine. This increase in stereospecificity was largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged alpha-proton. A similar increase in the stereospecificity of the second-order exchange rates for alanine was also largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged alpha-proton of D-alanine. Adding DL-alpha-glycerol 3-phosphate produced an increase in the stereospecificity of the second-order exchange rate observed with alanine but no significant change in the stereospecificity of the first-order exchange rate with tryptophan. The alpha-subunits are shown to increase the exchange rates of the alpha-protons of L-alanine and L-tryptophan. We conclude that the contribution of the R-group of an amino acid to the stereospecificity of the exchange reactions of its alpha-proton can be similar to or larger than that of its alpha-carboxylate group. Possible mechanisms that could explain the stereospecificity of these exchange reactions are discussed.
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