Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli - A study of structure and function
O. Fjellstrom et al., Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli - A study of structure and function, J BIOL CHEM, 274(10), 1999, pp. 6350-6359
Conformational changes in proton pumping transhydrogenases have been sugges
ted to be dependent on binding of NADP(H) and the redox state of this subst
rate. Based on a detailed amino acid sequence analysis, it is argued that a
classical beta alpha beta alpha beta dinucleotide binding fold is responsi
ble 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 cys
teine mutants (cf beta A348C, cf beta A390C, cf beta K424C, and cf beta RA2
5C) were introduced into a functional cysteine-free transhydrogenase. Also,
five cysteine mutants were constructed in the isolated domain III of Esche
richia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392
C, and ecIIIK424C). In addition to kinetic characterizations, effects of su
lfhydryl-specific labeling with N-ethylmaleimide, 2-(4'-maleimidylanilino)n
aphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleo
tide (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(4
24) are located in or close to the NADP(H) site. More specifically, beta-Al
a(348) succeeds beta B. The remarkable reactivity of beta R350C toward NNAD
P 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 substr
ate affinity,but it could also have a more complex role in the coupling mec
hanism for transhydrogenase.