MUTATION OF AMINO-ACID-RESIDUES IN THE MOBILE LOOP REGION OF THE NAD(H)-BINDING DOMAIN OF PROTON-TRANSLOCATING TRANSHYDROGENASE

The effects of single amino acid substitutions in the mobile loop regi on of the recombinant NAD(H)-binding domain (dI) of transhydrogenase h ave been examined. The mutations lead to clear assignments of well-def ined resonances in one-dimensional H-1-NMR spectra. As with the wild-t ype protein, addition of NADH, or higher concentrations of NAD(+), led to broadening and some shifting of the well-defined resonances. With many of the mutant dI proteins more nucleotide was required for these effects than with wild-type protein. Binding constants of the mutant p roteins for NADH were determined by equilibrium dialysis and, where po ssible, by NMR. Generally, amino acid changes in the mobile loop regio n gave rise to a 2-4-fold increase in the dI-nucleotide dissociation c onstants, but substitution of Ala(236) for Gly had a 10-fold effect. T he mutant dI proteins were reconstituted with dI-depleted bacterial me mbranes with apparent docking affinities that were indistinguishable f rom that of wild-type protein. In the reconstituted system, most of th e mutants were more inhibited in their capacity to perform cyclic tran shydrogenation (reduction of acetyl pyridine adenine dinucleotide, AcP dAD(+), by NADH in the presence of NADP(+)) than in either the simple reduction of AcPdAD(+) by NADPH, or the light-driven reduction of thio -NADP(+) by NADH, which suggests that they are impaired at the hydride transfer step. A cross-peak in the H-1-H-1 nuclear Overhauser enhance ment spectrum of a mixture of wild-type dI and NADH was assigned to an interaction between the A8 proton of the nucleotide and the beta CH3 protons of Ala(236). It is proposed that, following nucleotide binding , the mobile loop folds down on to the surface of the dI protein, and that contacts, especially from Tyr(235) in, Gly-Tyr-Ala motif with the adenosine moiety of the nucleotide, set the position of the nicotinam ide ring of NADH close to that of NADP(+) in dIII to effect direct hyd ride transfer. (C) 1998 Elsevier Science B.V. All rights reserved.