Interaction between Adenosine Triphosphate and Glyceraldehyde 3-Phosphate Dehydrogenase

Abstract

The inhibitory effect of ATP on glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) has been investigated under conditions simulating the intracellular milieu. The inhibition is strongly pH-dependent. With the physiological concentrations of ATP, NAD, and orthophosphate, the dehydrogenase reaction is 87 % inhibited at pH 6.8, 67 % at pH 7.4, and only 20 % at pH 8.1. ADP, adenosine 5′-monophosphate, and cyclic adenosine 3′:5′-monophosphate are almost equally inhibitory but have less physiological significance since the intracellular levels are much lower than ATP. The ATP inhibition is mixed type inhibition in which ATP is nearly competitive with respect to either NAD or Pi. ATP (6 mm) increases Km for NAD from 0.06 to 0.3 mm and decreases Vmax from 91 to 65 µmoles of NADH per min per mg of protein. Similarly the Km for Pi is changed from 1.2 to 6.1 mm whereas Vmax is decreased from 89 to 61. Experiments using AMP or pyrophosphate as the component parts of ATP have also been carried out. AMP reacts competitively with respect to NAD but not Pi, whereas PPi is nearly competitive with Pi but not NAD. These data suggest that ATP simultaneously obstructs both NAD and Pi binding in the active center. ATP is noncompetitive with regard to glyceraldehyde 3-phosphate. Mg2+ or Mn2+ reduces the inhibitory effect of ATP by complexing with the nucleotide. ATP also effects a time-dependent inactivation, which is additive with the instantaneous inhibition mentioned above. This time-dependent inhibition is counteracted by NAD but not Pi. The physiological implication of ATP inhibition on the dehydrogenase is discussed in the connection with muscle physiology. ATP inhibition may in part account for the high levels of glyceraldehyde 3-phosphate dehydrogenase in the muscle, protect against excessive acidity due to lactic production, limit maximal glycolytic flux, and modify the turnover of the enzyme.