DOMAINS, SPECIFIC RESIDUES AND CONFORMATIONAL STATES INVOLVED IN HYDRIDE ION TRANSFER AND PROTON-PUMPING BY NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE FROM ESCHERICHIA-COLI

Nicotinamide nucleotide transhydrogenase constitutes a proton pump whi ch Links the NAD(PI) and NADP(H) pools in the cell by catalyzing a rev ersible reduction of NADP(+) by NADH. The recent cloning and character ization of several proton-pumping transhydrogenases show that they sha re a number of features. They are composed of three domains, i.e., the hydrophilic domains I and III containing the NAD(H)- and NADP(H)-bind ing sites, respectively, and domain II containing the transmembrane an d proton-conducting region. When expressed separately, the two hydroph ilic domains interact directly and catalyze hydride transfer reactions similar to those catalyzed by the wild-type enzyme. An extensive muta genesis program has established several amino acid residues as importa nt for both catalysis and proton pumping. Conformational changes media ting the redox-driven proton pumping by the enzyme are being character ized With the cloned, well-characterized and easily accessible transhy drogenases from E. coli and Rhodospirillum rubrum at hand, the overall aim of the transhydrogenase research, the understanding of the confor mationally driven proton pumping mechanism, is within reach. (C) 1998 Elsevier Science B.V.