Reversible inhibition by pyridoxal 5'-phosphate and irreversible inactivation by urea and guanidine hydrochloride of thymidylate synthase from Lactobacillus leichmannii.

Pyridoxal 5'-phosphate reversibly inhibited thymidylate synthase from Lactobacillus leichmannii. The inhibition was competitive with dUMP (Ki = 1 microM) and non-competitive with 5,10-CH2-THF (Ki = 0.08 microM). Treatment of native or pCMB-treated enzyme with urea (5 M) or guanidine hydrochloride (4 M) resulted in inactivation and dissociation of the homodimer (74 kDa) into monomer (37 kDa).

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)