9513814 Levy Glucose-6-phosphate dehydrogenases (G6PDs) from different organisms differ with respect to their coenzyme specificity in vivo. Most, including human G6PD (hG6PD), are specific for NADP; a few, including G6PD from the bacterium Leuconostoc mesenteroides (LmG6PD), can utilize either NADP or NAD. This proposal seeks to elucidate differences among the coenzyme binding sites of these G6PDs with different coenzyme specificities. The investigators intend to identify critical amino acid residues in LmG6PD and the roles they play in differential binding of NADP and NAD, binding of G6P, and catalysis. All G6PDs consist of 2 or 4 identical subunits. The projected studies will also identify amino acids involved in interactions between the subunits of LmG6PD, and assess the role of intersubunit communication in this enzyme's functions. Steroids inhibit G6PD uncompetitively with respect to both coenzyme and substrate by binding to the ternary enzyme-NADP-subtrate complex. The proposal also seeks to identify the steroid binding site in hG6PD. The principal techniques to be used in identifying the amino acids responsible for these various properties are: a) site-directed mutagenesis, b) kinetic analyses and determination of binding constants on the purified mutant and wildtype enzymes, and c) X-ray crystallography. The proposed research will lead to an understanding of how the same enzyme (G6PD) catalyzing the same reaction (oxidation of glucose-6-phosphate) has evolved in different organisms (L. mesenteroides, and humans) to exhibit differences in coenzyme specificity and susceptibility to steroid inhibition, and precisely what protein structural features determine these differences. It will also provide a detailed description of the catalytic mechanism of G6PD, and identify those amino acids that are responsible for catalysis, binding of coenzyme and substrate, and transmitting information between individual enzyme subunits. %%% Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme in t he metabolism of carbohydrates and fats. It functions in the synthesis of two key metabolites: pentose sugars, required for the synthesis of nucleic acids, and NADPH, required for the biosynthesis of fats. This project seeks to elucidate which amino acids of G6PD from the bacterium Leuconostoc mesenteroides interact with coenzymes and substrate. The bacterial G6PD is used because it is a more stable and simpler enzyme, and because we have solved its three-dimensional structure. Also, unlike human G6PD, it can interact with two coenzymes (NADP and NAD) and how this occurs is, in itself, of great interest since most dehydrogenases are specific for only one coenzyme. We will also determine which human G6PD amino acids interact with inhibitory steroids. The principal technique to be used is site-directed mutagenesis, which allows us to replace any G6PD amino acid by any other one. By examining the kinetic and other properties of the mutant G6PD we can deduce the function of the amino acid that was replaced. ***
