Purification and Properties of the Pyridine Nucleotide Transhydrogenase from Pseudomonas aeruginosa

A 2'-AMP-activated pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa has been purified to apparent homogeneity and crystallized. The enzyme has a typical flavoprotein absorption spectrum. The spectrum is partially bleached by the anaerobic addition of TPNH and the bleaching is accompanied by increases in longer wave length absorption. Subsequent addition of DPN+ reverses the bleaching. The enzyme catalyzes reduction of dichloroindolphenol and potassium ferricyanide by TPNH and DPNH, and this reduction by DPNH is activated by 2'-AMP. If sedimentation in the analytical ultracentrifuge is monitored with schlieren optics or with a spectrophotometric scanning device measuring flavin absorption, a heterogeneous, aggregated system in the absence and an apparently homogenous system in the presence of 2'-AMP or TPN+ are observed. Incubation of the enzyme at 51° in the presence of TPNH or treatment of the enzyme with acidic ammonium sulfate causes a loss of activity. Addition of FAD, but not flavin mononucleotide, restores activity. The above evidence indicates that the transhydrogenase is a flavoprotein and suggests that the natural cofactor is FAD. The sedimentation velocity experiments indicate that under the conditions of observation the purified enzyme is very large. At a concentration of 8 mg per ml and in the presence of 2'-AMP or TPN+ the transhydrogenase sediments with an s20,w of 33.8. p-Hydroxymercuribenzoate can cause both inhibition and activation of the enzyme and protection against these effects is afforded by substrates. 2'-AMP activates several reactions in which the reducing substrate does not contain a 2'-phosphate group. When TPNH at about 3 x 10-4 m is the reducing substrate there is no activation by 2'-AMP. 2'-AMP enhances the rate of anaerobic reduction of enzyme-bound flavin by DPNH. This effect was observed at high concentrations of enzyme and may reflect the ability of 2'-AMP to disaggregate the enzyme, yet not necessarily reflect the mechanism by which 2'-AMP activates reactions in which only catalytic concentrations of enzyme are present. The enzyme has been tested for its ability to catalyze reduction or oxidation of a large number of physiological, nonpyridine nucleotide substrates. All substrates tested were inactive.