SUBSTRATE DETERMINANTS OF THE COURSE OF TARTRATE DEHYDROGENASE-CATALYZED REACTIONS

The substrate specificity of tartrate dehydrogenase has been probed us ing a series of alternative substrates to identify the molecular inter actions which determine whether a particular substrate undergoes enzym e-catalyzed decarboxylation or not. A series of 3-substituted malate a nalogs, in which F, Cl, Br, I, SH, or NH2 substituents were placed at the 3R- or 3S-position, was prepared, and the product resulting from t he action of tartrate dehydrogenase on each compound was identified. A ll of the halomalates and both diastereomers of aminomalate underwent oxidative decarboxylation; both diastereomers of 3-thiomalate underwen t net nonoxidative decarboxylation, The results were interpreted in te rms of a model in which decarboxylation is conformationally controlled . The data are not consistent with a model which suggests that substra tes assume the conformation that is necessary to avoid steric crowding between the enzyme and the substituent at the 3-position of the subst rate. These data are consistent with a model in which the course of th e reaction with (+)-tartrate and meso-tartrate is dictated by the coor dination of the substrate hydroxyls to the active site Mn2+. However, the observed reactivities of the 3-methyltartrate diastereomers are no t consistent with this model, either: (2R,3R)-3-methyltartrate undergo es oxidative decarboxylation, and (2R,3S)-3-methyltartrate undergoes s imple oxidation. These results suggest that for these compounds the co nformation is dictated by the positioning of the hydrophobic substitue nt in a specific binding pocket. In fact, the similarity in the K-m va lues for all the (2R,3S)-3-substituted substrates suggest that they sh are a common binding mode; the K-m's for the (2R,3R)-3-substituted sub strates are much higher and vary widely, suggesting that they bind in an alternative mode.