PMID: 9182734Jun 1, 1997Paper

Properties of a cysteine-free proton-pumping nicotinamide nucleotide transhydrogenase

The Biochemical Journal
J MeullerJ Rydström

Abstract

Nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated with respect to the roles of its cysteine residues. This enzyme contains seven cysteines, of which five are located in the alpha subunit and two are in the beta subunit. All cysteines were replaced by site-directed mutagenesis. The final construct (alphaC292T, alphaC339T, alphaC395S, alphaC397T, alphaC435S, betaC147S, betaC260S) was inserted normally in the membrane and underwent the normal NADPH-dependent conformational change of the beta subunit to a trypsin-sensitive state. Reduction of NADP+ by NADH driven by ATP hydrolysis or respiration was between 32% and 65% of the corresponding wild-type activities. Likewise, the catalytic and proton pumping activities of the purified cysteine-free enzyme were at least 30% of the purified wild-type enzyme activities. The H+/H- ratio for both enzymes was 0.5, although the cysteine-free enzyme appeared to be more stable than the wild-type enzyme in proteoliposomes. No bound NADP(H) was detected in the enzymes. Modification of transhydrogenase by diethyl pyrocarbonate and the subsequent inhibition of the enzyme were unaffected by removal of the cysteines, indicating a lack of involvement of cysteines in this p...Continue Reading

Citations

Jun 26, 1999·The Journal of Biological Chemistry·J Meuller, J Rydström
Oct 31, 2008·Journal of Bioenergetics and Biomembranes·Anders PedersenJan Rydström
Aug 12, 2020·Archives of Biochemistry and Biophysics·Rafaela BicegoRoger F Castilho
Sep 28, 2001·Biochemical and Biophysical Research Communications·A BerheB L Persson

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