Kinetics of inactivation and molecular asymmetry of NAD-specific glyceraldehyde-3-phosphate dehydrogenase of Pisum sativum.

Glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been purified to homogeneity from pea seeds. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis (with and without sodium dodecyl sulfate; subunit molecular weight 38 000). It is free from bound nucleotides. The kinetics of heat inactivation of the crude enzyme extract as well as the purified enzyme are biphasic, in that exactly half of the activity is destroyed more rapidly than the residual half. The data are consistent with the rate equation: (formula: (see text): where A0 and A are activities at times zero and t, respectively, and k1 and k2 are first-order rate constants for the fast and slow phases, respectively. Addition of NAD+ slows down thermal inactivation, without altering the overall kinetic pattern. The activity lost due to the fast component (k1) of the reaction is regained on colling ('annealing'), whereas the slow reaction (k2) is not reversed, suggesting the following scheme: formula (see text): This is confirmed by plotting the activity after 'annealing' against initial period of heating. A single first-order rate constant (k2) is observed. The enzyme possesses about one reactive SH group per subunit which can be titrated with 5,5'-dithio-bis-(2-nitrobenzoic acid). Blocking of these groups inactivates the enzyme. Inactivation with 20 micrometer N-ethylmaleimide and 30 micrometer iodoacetate (at pH 8.6 and 33 degrees C) follows simple first-order kinetics (rate constants 0.099 and 0.139 min-1, respectively), suggesting that all SH groups react equally readily with these reagents. Reaction of the enzyme with 0.6 micrometer p-chloromercuri benzoate, however, shows biphasic kinetics similar to thermal inactivation. The reaction of p-chloromercuri benzoate with partially heat-inactivated enzyme (residual activity 37.5%) follows simple first-order kinetics. The molecular asymmetry demonstrated by these results must arise from the unique quaternary structure of the enzyme molecule, which is apparently made up of chemically identical subunits (pseudo-isologous association).