INACTIVATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE WITH SH-REAGENTS AND ITS RELATIONSHIP TO THE PROTEIN QUATERNARY STRUCTURE

Inactivation of mung bean glyceraldehyde-3-phosphate dehydrogenase (GP DH) with excess iodoacetate or N-ethylmaleimide exhibits pseudo-first order kinetics at pH 7.3 and 8.6 in the absence and presence of NAD+, suggesting that all the reactive SH groups (four per tetrameric GPDH m olecule) have equivalent reactivity towards these reagents. This is si milar to the D2-symmetry conformation proposed on the basis of thermal inactivation data [Malhotra and Srinivasan, Arch. Biochem. Biophys. 2 36, 775-781 (1985)]. With p-chloromercury benzoate (p-CMB), the inacti vation of GPDH is very fast and its kinetics can be monitored at low r eagent concentration only. Keeping a high molar p-CMB: enzyme ratio (= 47), the kinetics were found to be biphasic, with half of the activit y being lost in a fast and the remaining in a slow phase, characterist ic of C2-symmetry conformation and half site reactivity. The p-CMB ina ctivation could be largely reversed on the addition of excess cysteine . A comparison of these data with literature reports on this and other GPDHs reveals that all reagents having large non-polar moieties exhib it half site reactivity with this enzyme.