Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli -: A study of structure and function

Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical beta alpha beta alpha beta dinucleotide binding fold is responsible for binding NADP(H). A model defining beta A, alpha B, beta B, beta D, and beta E of this domain is presented. To test this model, four single cysteine mutants (cf beta A348C, cf beta A390C, cf beta K424C, and cf beta RA25C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined. The results are consistent with the view that, in agreement with the model, beta-Ala(348), beta-Arg(350), beta-Ala(390), beta-Asp(392), and beta-Lys(424) are located in or close to the NADP(H) site. More specifically, beta-Ala(348) succeeds beta B. The remarkable reactivity of beta R350C toward NNADP suggests that this residue is close to the nicotinamide moiety of NADP(H). beta-Ala(390) and beta-Asp(392) terminate or succeed beta D, and are thus, together with the region following PA, creating the switch point crevice where NADP(H) binds. beta-Asp(392) is particularly important for the substrate affinity,but it could also have a more complex role in the coupling mechanism for transhydrogenase.