The polymeric enzyme transhydrogenase from Pseudomonas aeruginosa (NADPH:NAD+ oxidoreductase; EC 1.6.1.1) has been shown to possess distinct catalytic and regulatory sites, despite the close structural relationship between substrates and effectors. The present report substantiates the previous conclusions from kinetic and affinity chromatography studies, which have suggested that the substrates NADPH and oxidized thionicotinamide ademine dinucleotide phosphate could bind to both catalytic and regulatory sites. In addition, the allosteric R form of the enzyme appears now to be stabilized against high dilution inactivation.
The oxidized substrate thionicotinamide adenine dinucleotide forms a dead end complex on binding to the catalytic site in the T form. The process is slow, and can be termed hysteretic, as defined by Frieden (J. Biol. Chem. (1970), 245, 5788-5799).