PROPERTIES OF A CYSTEINE-FREE PROTON-PUMPING NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE

Nicotinamide nucleotide transhydrogenase from Escherichia coli was inv estigated with respect to the roles of its cysteine residues. This enz yme contains seven cysteines, of which live are located in the alpha s ubunit and two are in the beta subunit. All cysteines were replaced by site-directed mutagenesis. The final construct (alpha C292T, alpha C3 39T, alpha C395S, alpha C397T, alpha C435S, beta C147S, beta C260S) wa s inserted normally in the membrane and underwent the normal NADPH-dep endent conformational change of the beta subunit to a trypsin-sensitiv e state. Reduction of NADP(+) by NADH driven by ATP hydrolysis or resp iration was between 32%, and 65%, of the corresponding wild-type activ ities. Likewise, the catalytic and proton pumping activities of the pu rified cysteine-free enzyme were at least 30% of the purified wild-typ e enzyme activities. The H+/H- ratio for both enzymes was 0.5, althoug h the cysteine-free enzyme appeared to be more stable than the wild-ty pe enzyme in proteoliposomes. No bound NADP(H) was detected in the enz ymes. Modification of transhydrogenase by diethyl pyrocarbonate and th e subsequent inhibition of the enzyme were unaffected by removal of th e cysteines, indicating a lack of involvement of cysteines in this pro cess. Replacement of cysteine residues in the a subunit resulted in no or little change inactivity, suggesting that the basis for the decrea sed activity was probably the modification of the conserved beta-submi t residue Cys-260 or (less likely) the non-conserved beta-subunit resi due Cys-147. It is concluded that the cysteine-free transhydrogenase i s structually and mechanistically very similar to the wild-type enzyme , with minor modifications of the properties of the NADP(H) site, poss ibly mediated by the beta C260S mutation. The cysteine-free construct will be a valuable tool for studying structure-function relationships of transhydrogenases.