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Phycomyces blakesleeanus glutathione reductase shows hysteretic behaviour under experimental conditions, when GSSG substrate inhibition is observed. The progress curves for the reaction show an acceleration phase. The degree of hysteresis varied inversely as the enzyme concentration. It increased when GSSG or NADPH concentration increased, whereas the addition of GSH or NADP+ to the initial reaction mixture prevented it from occurring. In addition, hysteresis was dependent on pH, ionic strength and temperature, decreasing as any of these parameters increased. The parallel effects of pH and ionic strength on the GSSG substrate inhibition and hysteretic behaviour suggest a relationship between these two mechanisms. From the overall results reported in this paper, we propose that the hysteretic behaviour shown by Phycomyces glutathione reductase could be due to a process of time-dependent accumulation of reaction products rather than to a slow conformational change.

Studies on ATP effects on the allosteric kinetics shown by pyruvate kinase from Phycomyces blakesleeanus NRRL 1555 (-) are reported. Phosphoenolpyruvate showed an allosteric ATP-dependent substrate inhibition. The results supported the existence of spatially distinct catalytic binding sites and the inhibitory binding sites for phosphoenolpyruvate, and ATP showed opposite heterotropic effects with respect to these two types of binding site. With respect to Mg2+ ions, ATP caused a negative heterotropic effect. The global inhibitory effect of ATP was in agreement with the predictions postulated by the two-state concerted-symmetry model of Monod, Wyman and Changeux.

We have partially characterized an intracellular fraction from Phycomyces blakesleeanus which shows proteolytic activity. The apparent thermal inactivation constant (Kd) was 0.12 min-1 at 50 degrees C. This proteolytic fraction was split into two active fractions by ultrafiltration using a membrane with an exclusion size of 30,000. Both fractions were inhibited by phenyl methyl sulphonyl fluoride. The Ki value for the fraction with molecular weight greater than 30,000 was 0.075 mM. The fraction with molecular weight less than 30,000 inactivated the Phycomyces CPS.