Glyoxalase I (Glx. I) and formaldehyde dehydrogenase (FDH) are two glutathione (GSH)-dependent enzymes that operate on an equilibrium mixture of potential substrate forms composed of free aldehyde, GSH and the corresponding thiohemiacetal adduct. The overall objective of the proposed research is to determine the catalytic significance and molecular basis of the substrate specificities of these enzymes. In order to achieve this objective, the following experiments are proposed that are based, in part, on the work of the last two years. First, the nonenzymic rates of interconversion of the diasteriomers due to the physiologically important methylglyoxal-GSH thiohemiacetal will be determined by C-13 selective-inversion-recovery NMR methods. This is a follow up experiment based on the observation that the corresponding interconversion rate of the diasteriomers due to phenylglyoxal-GSH thiohemiacetal are slow (k = 10 sec-1, pH 7) in comparison to the catalytic turnover number of G1x. I. (about 500 sec-1, pH 7). This may explain the evolved capacity of the enzyme to use both diasteriomers as substrates. Second, G1x. I catalyzed interconversion of the diasteriomers due to the nonsubstrate, glyoxylic acid-GSH thiohemiacetal, will be tested for on the basis of NMR methods. A positive indication of such a catalyzed process has been obtained on the basis of a preliminary NMR line-broadening analysis of the diasteriomers in the presence of enzyme. This observation is indicative of enzyme catalyzed interconversion of the bound substrate diasteriomers before transformation to bound product. Third, the overall stereochemistry of G1x. I catalyzed interconversion of S-(D)-dithiolactoyl glutathione to the corresponding, exchange inert dithiohemiacetals will be determined in order to establish the stereochemistry of the enediol intermediate on the reaction pathway. Fourth, the ability of G1x. I to discriminate between the diasteriomers due to thiohemiacetals, formed between Alpha-ketoaldehydes and sterically hindered derivatives of GSH, will be obtained as a test of the hypothesis that positional mobility of the glutathionyl sulfur of bound substrate is required in order for the enzyme to use both diasteriomers of the normal substrate thiohemiacetals. Finally, isozymes of FDH will be tested for as a follow on preliminary observations. Contrary to previous reports, methylglyoxal is not a substrate for FDH.
