Protective effect of the pteroylpolyglutamates and phosphate on the proteolytic inactivation of thymidylate synthetase

Thymidylate synthetase is readily inactivated by trypsin, chymotrypsin, and carboxypeptidase A when incubated in 10–20 mm potassium phosphate buffer (pH 7.0). The loss is activity produced by trypsin and chymotrypsin is accomplished by extensive protein degradation, while inactivation by carboxypeptidase A is accompanied by release of the carboxyl-terminal valine only (Aull et al., 1974, J. Biol. Chem., 249, 1167–1172). In contrast, when the incubations are conducted in 100–200 mm potassium phosphate buffer (pH 7.0), the synthetase is not inactivated by any of the three enzymes and the results of amino acid analysis and sodium dodecyl sulfate disc gel electrophoresis demonstrate that proteolysis is prevented. The resistance of thymidylate synthetase to inactivation was shown not to be due to the inhibition of the proteolytic enzymes by the buffer. The inactivation is not prevented either by pteroylmonoglutamates or by 2′-deoxyuridine 5′-phosphate (dUMP) alone, but the presence of both is partially protective. The pteroylpolyglutamates, however, offer limited protection against carboxypeptidase A and chymotrypsin; in combination with dUMP, proteolytic inactivation of the snythetase by all three enzymes is prevented. Characterization of the properties of carboxypeptidase A-inactivated thymidylate synthetase reveals the following, (i) The binding of deoxynucleotides is unaltered, but the binding of a variety of pteroylpolyglutamate derivatives is reduced or abolished, (ii) Pteroylpolyglutamates are bound provided dUMP or an analog such as 5-fluorodUMP is present, (iii) Ternary complex formation between carboxypeptidase A-inactivated enzyme and (+)5,10-methylenetetrahydropteroyltetraglutamate plus 5-fluorodUMP occurs in the same molar binding ratio (1:2:2) at saturation as with the native enzyme, but differs from the native enzyme ternary complex in that the dissociation constant for 5-fluorodUMP is increased by approximately 10⁵. In addition, there is no evidence for the formation of covalent linkages between the ligands and enzyme, (iv) The treated enzyme cannot catalyze tritium release from ³H₅]dUMP in the presence of either (+)5,10-methylenepteroylmonoglutamate or (+)5,10-methylenetetrahydropteroyltetraglutamate.