The glucose-6-phosphate dehydrogenases of L. mesenteroides and Z. mobilis exhibit dual coenzyme specificities, utilizing either NAD or NADP. The long-range goal of these studies is to develop an understanding of the structural features of these enzymes which permit this dual specificity. Site-directed mutagenesis will be utilized in order to identify those residues involved in specificity for each coenzyme and perhaps to switch specificities. Chemical modification studies will be utilized to identify coenzyme binding sites. Kinetic studies will be employed to characterize the enzyme in the presence of different coenzymes. The conformation change induced by the coenzymes will be characterized. These studies will provide new insights into the relationship between structure and function in the more common NAD- or NADP- specific glucose-6-phosphate dehydrogenases. %%% Enzymes involved in metabolic pathways exhibit high specificity for coenzymes. In fact, this dependence on the presence of specific coenzymes is often utilized in the general control of metabolic pathways. What structural aspects of the glucose-6-phosphate dehydrogenases (G6PDs) determine their requirement for NAD or NADP coenzymes is an interesting and important question. The proposed research utilizes two interesting G6PDs which show dual coenzyme specificity. Binding of either NAD or NADP induces structural changes in the enzyme which result in quite distinct catalytic properties. Site-directed mutagenesis will be utilized to probe the structure of the enzyme as it relates to coenzyme binding. The results will provide insights into the functional behavior of the more common NAD- or NADP-specific G6PDs.
