Fast hydride transfer in proton-translocating transhydrogenase revealed in a rapid mixing continuous flow device.
Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Coupling is achieved through changes in protein conformation. Upon mixing, the isolated nucleotide-binding components of transhydrogenase (dI, which binds NAD(H), and dIII, which binds NADP(H)) form a catalytic dI(2).dIII(1) complex, the structure of which was recently solved by x-ray crystallography. The fluorescence from an engineered Trp in dIII changes when bound NADP(+) is reduced. Using a continuous flow device, we have measured the Trp fluorescence change when dI(2).dIII(1) complexes catalyze reduction of NADP(+) by NADH on a sub-millisecond scale. At elevated NADH concentrations, the first-order rate constant of the reaction approaches 21,200 s(-1), which is larger than that measured for redox reactions of nicotinamide nucleotides in other, soluble enzymes. Rather high concentrations of NADH are required to saturate the reaction. The deuterium isotope effect is small. Comparison with the rate of the reverse reaction (oxidation of NADPH by NAD(+)) reveals that the equilibrium constant for the redox reaction on the complex is >36. This high value might be important in ensuring high turnover rates in the intact enzyme.
Pinheiro
T J
TJ
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Venning
J D
JD
Jackson
J B
JB
D018360:Crystallography, X-Ray; D019281:Dimerization; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D012246:Rhodospirillum / Q000201:enzymology; D013997:Time Factors
D009711:Nucleotides; D011522:Protons; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D009250:NADP Transhydrogenases