Reassociation and Reactivation of Yeast Glyceraldehyde‐3‐Phosphate Dehydrogenase after Dissociation in the Presence of ATP

Abstract

Tetrameric d ‐glyceraldehyde‐3‐phophate dehydrogenase from yeast in the presence of 5 mM ATP and 0.1 M 2‐mercaptoethanol at pH 8.5 can be reversibly inactivated and dissocited by changing the temperature from 25 to 0°C. Using ultracentrifugation and gel chromatography, the inactive low‐temperature dissociation product is found to be a compact monomer ( M r = 36000, s 20,w = 3.2S) which is still capable of binding on molecule of NAD + per subunit. Deactivation caused by dissociation at 0°C is a slow process with a first‐order rate constant of k = 0.007 min −1 (τ 1/2 = 180 min); it follows the instantaneous competitive inhibition of the ezyme by ATP which is characterized by K i = 2.3 mM. Reactivation at high enzyme concentration ( c > 10 μg/ml) obeys first‐order kineics. At low enzyme concentration a shift to a higher reaction order is obseved. Alimiting value of n = 2 is reached at 0.9 μg/ml. The data are in accordance with a uni‐bi molecular mechanism involving the association of (inactive) monomers as a prerequisite of enzymatic of enzymatic activity. The kinetic constants. k 1 = 9 ± 2 × 10 −1 and k 2 = 7 ± 1 × 10 4 M −1 × s −1 , correspond to previous data reported for the reactivation after full denaturation and dissociation of the enzyme in 6 M guanidine · HCI at pH 2.3 [Rudolph et al. Eur. J. Biochem. 81 , 563–570 (1977)]. This suggests a common intermediate in the reactivation process after deactivation and disscoiation under widely differing conditions.