Regulatory Mechanisms Involving Nicotinamide Adenine Nucleotides As Allosteric Effectors

Glucose 6-phosphate dehydrogenase from Escherichia coli is purified about 170-fold. The purified enzyme has no absolute requirement for divalent metal ions for activity. However, it is activated by Mg++, spermidine, or a number of other polycations. The apparent activation constant for spermidine is approximately 0.2 mm. In the presence of saturating concentrations of spermidine or other polycations, Mg++ ions have no further stimulatory effect. The enzyme is inhibited in an allosteric manner by DPNH and this inhibition is not reversed by DPN+, AMP, or spermidine. Compounds of physiological interest related to DPNH (ATP, GTP, acetyl coenzyme A, and coenzyme A) do not cause any inhibition. The initial velocity plots of the enzyme follow the Michaelis-Menten equation in the absence of DPNH. In its presence, however, the velocity versus substrate plots for TPN+ become sigmoidal (but remain hyperbolic for glucose-6-P as the variable substrate). Inhibition against both of the substrates of the enzyme by DPNH is noncompetitive. In addition, the inhibition curves for DPNH are also sigmoidal, suggesting a multisite binding of the inhibitor on the enzyme surface. A comparison is made between the kinetic characteristics of glucose-6-P dehydrogenase and different enzymes of carbohydrate metabolism in E. coli which have been demonstrated to be inhibited by DPNH, and it is pointed out that they all have one property in common, viz. that the rate-concentration plots for only one of the reactant substrates are sigmoidal in the presence of the inhibitor. The physiological implications of such kinetic behavior are discussed and it is concluded that the oxidative pentose pathway serves primarily to generate reducing power (TPNH) for biosynthetic purposes.