The long term objective is to elucidate the mechanisms by which binding energy in enzyme-substrate complexes facilitates enzymatic catalysis. A global approach will be adopted in coordinated studies of four enzymes that participate in carbohydrate metabolism and phosphotransfer, UDP- galactose 4-epimerase (Ga1E), galactose-1-phosphate uridlyltransferase (Ga1T), galactokinase (Ga1K), dTDP-glucose 4,6-dehydratase (dehydratase) and human Fhit, a putative tumor suppressor. Chemical, kinetic, spectroscopic, mutagenic, and crystallographic methods will be employed. Galactose metabolism is essential in all living cells and presents fundamental questions bearing on binding energy and catalysis. Cells must break galactose down for use as a fuel to produce galactose for glycoconjugates. One manifestation of the importance of galactose metabolism is the metabolic defect underlying galactosemia, in which galactose metabolism is impaired by defects in GalT. Research on three enzymes of galactose metabolism will be emphasized. The enzymes from E. coli will be studied as models for the mammalian enzymes, which are homologous. The principle objective in studies of Ga1E will be to describe the molecular basis for the enhancement of the chemical reactivity of the niacin- co-enzyme NAD+ by the use of binding energy between the UDP-moiety of substrates and the enzyme. Another Ga1E objective is to elucidate the mechanism of general acid/base catalysis, which appears to be carried out by tyrosine 149 and serine 124. The structural basis for a charge-transfer interaction between NAD+ and the enzyme will be determined. Research on dehydratase will be compared and contrast its biological mechanism with that of Ga1E. The objectives for research on Ga1T include the elucidation of how binding energy is used to stabilize the uridylyl-enzyme intermediate, how metal ions stabilize the active conformation, and how specific galactosemia mutations undermine the enzymatic activity. The standard free energy for the formation of the uridylyl-enzyme in site-directed mutant forms will be compared with wild-type enzyme to investigate binding interactions that stabilize the intermediate. Research on Fhit is directed toward elucidating its structural and mechanistic relationships with GalT. Research on the GalK will be initiated to determine its chemical mechanism and structure. The stereochemical course of phosphotransfer by GalK will be unmasked to determine whether a single or double- displacement mechanism is in full operation. Crystallization trials will be pursued with the objective of determining the first structured in the Ga1K family of sugar phosphotransferases.