Effects of polyoxyanions on sulfhydryl group modification of thymidylate synthetase

Essential arginyl residues in thymidylate synthetase from amethopterin-resistant Lactobacillus casei

“…Treatment of phenylglyoxal-modified enzyme, retaining 10% of its native activity, with [I4C]MMTS, in either 125 mM bicarbonate or 50 mM Mops buffer, pH 8.0, yielded 1.7 titratable sulfhydryl groups. The modification of 1.7 sulfhydryls is consistent with the previously reported results of Lewis et al (1978) and Plese & Dunlap (1977), in which the incorporation of radiolabel was correlated with loss of both enzymatic activity and the ability to form a ternary complex. Consequently, the formation of thiohemiacetals as the basis of inactivation by phenylglyoxal can be dismissed.…”

Fluorine-19 nuclear magnetic resonance investigation of the noncovalent and covalent binary complexes of 5-fluorodeoxyuridylate and Lactobacillus casei thymidylate synthetase

“…Methyl methanethiolsulfonate, MMTS, was employed to prepare thymidylate synthetase in which the catalytic cysteine sulfhydryls were blocked by a CH3Sgroup (Lewis et al, 1978b). Concentrated DEAE-enzyme was activated by dialysis in 50 mM Pipes buffer, pH 7.4, containing 1 mM EDTA and 25 mM 2-mercaptoethanol and then dethiolated on a Sephadex G-10 column (2 X 24 cm).…”

The effect of inorganic phosphate on the stability of some enzymes

“…In many cases P1 induces critical conformational changes in proteins; for example, the binding of Pi to glutamate dehydrogenase affects the reactivity of the thiol groups and the association-dissociation equilibrium, prevents or loosens the binding of guanosine triphosphate and decreases the Michaelis constants for the substrate and the coenzyme (for a review, see Sund et al, 1975). In other cases P; decreases the rate of enzyme inactivation (Rudolf & Jaenicke, 1976;Rippa et al, 1978;Sund et al, 1975;Lewis et al, 1978;Chlebowski et al, 1979;Torchilin et al, 1979;Holzman & Baldwin, 1980) and unfolding (Barnard, 1966) and affects the equilibrium between folded and unfolded forms of proteins (Barnard, 1966). Often these effects are given also by sulphate (Rippa et al, 1978;Lewis et al, 1978;Torchilin et al, 1979), Table 1.…”

“…In other cases P; decreases the rate of enzyme inactivation (Rudolf & Jaenicke, 1976;Rippa et al, 1978;Sund et al, 1975;Lewis et al, 1978;Chlebowski et al, 1979;Torchilin et al, 1979;Holzman & Baldwin, 1980) and unfolding (Barnard, 1966) and affects the equilibrium between folded and unfolded forms of proteins (Barnard, 1966). Often these effects are given also by sulphate (Rippa et al, 1978;Lewis et al, 1978;Torchilin et al, 1979), Table 1. Effect of P1 and sulphate on the rate of inactivation of 6-phosphogluconate dehydrogenase by some inactivating agents The values reported are periods of incubation in minutes required to obtain a 50% inactivation.…”