In mitochondria and bacteria, transhydrogenase uses the transmembrane proton gradient (δp) to drive reduction of NADP+ by NADH. We have investigated the pre-steady-state kinetics of NADP+ reduction by acetylpyridine adenine dinucleotide (AcPdADH, an analogue of NADH) in complexes formed from the two, separately prepared, recombinant, peripheral subunits of the enzyme: the dI component, which binds NAD+ and NADH, and the dIII component, which binds NADP+ and NADPH. In the stopped-flow spectrophotometer the reaction proceeds as a single-turnover burst of hydride transfer to NADP+ on dIII before product NADPH release becomes limiting in steady state. The burst is biphasic. The results indicate that the fast phase represents direct hydride transfer from AcPdADH to NADP+ in dI:dIII complexes, and that the slow phase, which predominates when [dI] < [dIII], corresponds to dissociation of the protein complexes during multiple turnovers of dI. Measurements on the amplitude of the burst, and on the apparent first-order rate constant of the fast phase, indicate that the equilibrium constant of the hydride-transfer step on the enzyme is shifted relative to that in solution. This has consequences for a model proposed earlier, in which δp is used, not at the hydride-transfer step, but to change the binding affinities of NADP+ and NADPH.
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Research Article|
July 08 1999
A shift in the equilibrium constant at the catalytic site of proton-translocating transhydrogenase: significance for a ‘binding-change’ mechanism
Jamie D. VENNING;
Jamie D. VENNING
1School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
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J. B JACKSON
J. B JACKSON
1
1School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
1To whom correspondence should be addressed (j.b.jackson@bham.ac.uk).
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Publisher: Portland Press Ltd
Received:
January 25 1999
Revision Received:
March 31 1999
Accepted:
May 06 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 341 (2): 329–337.
Article history
Received:
January 25 1999
Revision Received:
March 31 1999
Accepted:
May 06 1999
Citation
Jamie D. VENNING, J. B JACKSON; A shift in the equilibrium constant at the catalytic site of proton-translocating transhydrogenase: significance for a ‘binding-change’ mechanism. Biochem J 15 July 1999; 341 (2): 329–337. doi: https://doi.org/10.1042/bj3410329
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