A change in ionization of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase regulates both hydride transfer and nucleotide release.
Transhydrogenase couples the transfer of hydride-ion equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The enzyme has three components: dI binds NAD(H), dIII binds NADP(H) and dII spans the membrane. Coupling between transhydrogenation and proton translocation involves changes in the binding of NADP(H). Mixtures of isolated dI and dIII from Rhodospirillum rubrum transhydrogenase catalyse a rapid, single-turnover burst of hydride transfer between bound nucleotides; subsequent turnover is limited by NADP(H) release. Stopped-flow experiments showed that the rate of the hydride transfer step is decreased at low pH. Single Trp residues were introduced into dIII by site-directed mutagenesis. Two mutants with similar catalytic properties to those of the wild-type protein were selected for a study of nucleotide release. The way in which Trp fluorescence was affected by nucleotide occupancy of dIII was different in the two mutants, and hence two different procedures for determining the rate of nucleotide release were developed. The apparent first-order rate constants for NADP(+) release and NADPH release from isolated dIII increased dramatically at low pH. It is concluded that a single ionisable group in dIII controls both the rate of hydride transfer and the rate of nucleotide release. The properties of the protonated and unprotonated forms of dIII are consistent with those expected of intermediates in the NADP(H)-binding-change mechanism. The ionisable group might be a component of the proton-translocation pathway in the complete enzyme.
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
D J
DJ
Rodrigues
J D
JD
Venning
P G
PG
Quirk
J B
JB
Jackson
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D005453:Fluorescence; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009249:NADP / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits; D011522:Protons; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D009711:Nucleotides; D021122:Protein Subunits; D011522:Protons; D009249:NADP; D006859:Hydrogen; D014364:Tryptophan; D009250:NADP Transhydrogenases