Effect of substrate and phosphate ions on the quaternary structure symmetry of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Effects of glyceraldehyde-3-phosphate (G-3-P) and phosphate ions on thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases (GPDHs) of mung beans and rabbit muscle have been studied at different pH. In the absence of any ligand, the two enzymes show a striking similarity in the pH-dependence of the kinetics of thermal inactivation. At lower pH values both the enzymes biphasic kinetics with each phase accounting for about half of the starting activity (a C2 symmetry of the homotetrameric enzyme molecule). The kinetics change to a single exponential decay at higher pH values, a D2 symmetry [Malhotra & Srinivasan (1985) Arch. Biochem. Biphys. 236, 775-781; Malhotra & Tikoo (1991) Indian, J. Biochem. Biophys. 28, 16-21]. With each enzyme, phosphate ions are found to have no effect on the kinetic pattern at lower pH, but G-3-P brings about a change from biphasic to a single exponential decay. At higher pH values, G-3-P has no effect on the single exponential decay kinetic pattern, but phosphate ions change the same to a biphasic loss of activity with each phase accounting for about half of the starting activity. It has been concluded that with both the enzymes, G-3-P and phosphate ions have higher affinity and stabilise the D2- and C2-symmetry conformation, respectively. Binding isotherms of the two substrates for these enzymes have been described based on the ligand concentration-dependence of the changes in the rate constants and kinetic pattern of thermal inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of thermal inactivation and quaternary structure symmetry of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase.

Kinetics of thermal inactivation of apo-glyceraldehyde 3-phosphate dehydrogenase have been investigated under various conditions. At most pH values, the loss of enzyme activity takes place in two phases, a fast and a slow phase. The data correspond to the rate equation A = Afast.e-kfast.t + Aslow.e-kslow, where A is the observed residual activity (expressed as % of initial activity) at time t, Afast and Aslow are amplitudes (expressed as % of initial activity, so that Afast + Aslow = 100) and kfast and kslow the rate constants of the fast and slow phases, respectively. At pH 9 or below, Afast = Aslow = 50%. As pH is increased above 9, Afast increases gradually till at pH 10 or above when it accounts for the entire initial activity (single exponential decay). The rate constants of the two phases are strongly affected by the nature of the buffer, temperature and pH, but the amplitudes depend on pH alone. It has been suggested that the tetrameric enzyme exists in two conformations of different molecular symmetry, namely C2 (two pairs of sites of unequal stability, predominating at pH 9 or below) and D2 symmetry (four equivalent sites, predominating at pH 10 or above). The C2 in equilibrium D2 transformation is found to be highly cooperative with midpoint at pH 9.6.