title: Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme.
abstract: Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme. Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.
authors.affiliation: Department of Biochemistry and Biophysics, Göteborg University, Box 462, 405 30, Göteborg, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Pedersen
authors.forename: Jenny
authors.initials: J
authors.lastname: Karlsson
authors.forename: Magnus
authors.initials: M
authors.lastname: Althage
authors.forename: Jan
authors.initials: J
authors.lastname: Rydström
mesh_terms: D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017494:Proton Pumps / Q000737:chemistry / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D017494:Proton Pumps; D009243:NAD; D009249:NADP; D009250:NADP Transhydrogenases; D000469:Alkaline Phosphatase