High cyclic transhydrogenase activity catalyzed by expressed and reconstituted nucleotide-binding domains of Rhodospirillum rubrum transhydrogenase.
The hydrophilic, extramembranous domains I (alpha 1 subunit) and III of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase were expressed in Escherichia coli and purified therefrom as soluble proteins. These domains bind NAD(H) and NADP(H). respectively, and together they form the enzyme's catalytic site. We have demonstrated recently that the isolated domains I and III of the bovine transhydrogenase (or domain I of R. rubrum plus domain III of the bovine enzyme) reconstitute to catalyze transhydrogenation in the absence of the membrane-intercalated domain II, which carries the enzyme's proton channel. Here we show that the expressed domains I and III of the R. rubrum transhydrogenase catalyze a very high NADP(H)-dependent cyclic transhydrogenation from NADH to AcPyAD (3-acetylpyridine adenine dinucleotide) with a Vmax of 214 mumol AcPyAD reduced (min x mg of domain I)-1. The reaction mechanism is 'ping-pong' with respect to NADH and AcPyAD, as these nucleotides bind interchangeably to domain I, and the stereospecificity of hydride ion transfer is from the 4A position of NADH to the 4A position of AcPyAD. The expressed domain I is dimeric, like the native alpha 1 subunit of the enzyme, but the expressed domain III is monomeric and contains 0.94 mol NADP(H) per mol.
Yamaguchi
M
M
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037, USA.
Hatefi
Y
Y
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D008970:Molecular Weight; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; C005941:3-acetylpyridine adenine dinucleotide; D009250:NADP Transhydrogenases