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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

Artikel i vetenskaplig tidskrift
Författare Anders Pedersen
J Karlsson
Magnus Althage
Jan Rydström
Publicerad i Biochim.Biophys. Acta (Bioenergetics)
Volym 1604
Sidor 55-59
Publiceringsår 2003
Publicerad vid Institutionen för kemi
Sidor 55-59
Språk en
Länkar dx.doi.org/10.1016/S0005-2728(03)00...
Ämnesord Transhydrogenase; NAD; NADP; Proton pump; Membrane protein
Ämneskategorier Kemi

Sammanfattning

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 Kd 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.

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