Search

An investigation of the reaction mechanism of glutathione reductase isolated from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) was conducted. The enzyme showed GSSG concentration-dependent substrate inhibition by NADPH and pH-dependent substrate inhibition by GSSG. At pH 7.5, the kinetic data were consistent with a basic scheme corresponding to the branching mechanism, involving a ping-pong with formation of a dead-end F.NADPH complex and an ordered sequential mechanism. Both pathways have in common the step in which NADPH binds to the free oxidized form (E) of the glutathione reductase. At low concentrations of GSSG the ping-pong mechanism prevails, whereas at high concentrations the ordered mechanism appears to dominate. The data were analyzed on the basis of the limiting ping-pong mechanism with F.NADPH complex formation and of the hybrid mechanism, and the kinetic constants of the model were calculated. The data obtained at acidic pH values do not rule out the possibility that the kinetic model may be more complicated than the basic scheme studied.

The properties of ornithine decarboxylase from Phycomyces blakesleeanus were examined. Enzyme from mycelial cultures was extracted and purified approximately 70-fold. The apparent molecular weight is 96K. The Michaelis constants with respect to ornithine and pyridoxal 5'-phosphate are 90 and 0.37 microM, respectively. Putrescine is a potent competitive inhibitor with a Ki of 75 microM. Exposure of ornithine decarboxylase to sulfhydryl-modifying reagents resulted in a rapid inhibition of activity. In vivo addition of putrescine produced characteristic decreases in cellular ornithine decarboxylase activity. Light stimulation of dark-adapted mycelial cultures also decreased cellular ornithine decarboxylase activity.

Geranylgeranyl diphosphate synthase has been purified to homogeneity from the carotene-overproducing strain M1 of Phycomyces blakesleanus. Usually two activity peaks with molecular weights of 60,000 and 30,000 eluted on gel exclusion chromatography, suggesting that the enzyme consists of two subunits, with a tendency to dissociate. With homogeneous protein, a single-staining band with molecular weight of 30,000 appeared on sodium dodecyl sulfate gel electrophoresis, confirming a subunit molecular weight of 30,000. Only isopentenyl diphosphate and farnesyl diphosphate were accepted by this enzyme for geranylgeranyl diphosphate formation. The smaller allylic compounds, dimethylallyl and geranyl diphosphate, were utilized at less than 1/20th the rate of farnesyl diphosphate. Michaelis constants of 9 microM for isopentenyl diphosphate and 60 microM for farnesyl diphosphate were found. The isoelectric point is 4.8.