ESSENTIAL ARGINYL RESIDUES IN THYMIDYLATE SYNTHETASE FROM AMETHOPTERIN-RESISTANT LACTOBACILLUS-CASEI

Thymidylate synthetase from amethopterin-re-sistant Lactobacillus casei is rapidly and completely inactivated by phenylglyoxal, a reagent that is highly selective for the modification of arginyl residues. Both dUMP and dTMP afford significant protection, while 5, 10-methylenetetra-hydrofolate provides little protection against phenylglyoxal-dependent inactivation. Extrapolation to complete inactivation suggests that inactivation by phenylglyoxal correlates with the modification of 1.8 arginyl residues per enzyme subunit, as determined by the incorporation of [7-14C]phenylglyoxal. The presence of either dUMP or dTMP protects approximately 1.0 and 0.7 arginyl residues per enzyme subunit, respectively, against incorporation of [7-14C]phenylglyoxal. In a preliminary study [Cipollo, K. L„ & Dunlap, R. B. (1978) Biochem. Biophys. Res. Commun. 81, 1139-1144], it was reported that the enzyme is completely inactivated by 2, 3-butanedione in borate buffer. Results of amino acid analysis suggest that the complete loss of activity by 2, 3-butanedione correlates with the modification of 2.3 arginyl residues per subunit and that dUMP and FdUMP protect 0.7 and 1.1 arginyl residues per subunit, respectively. Similarly, in the ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate, and 5, 10-methylene-tetrahydrofolate, 1.1 arginines were protected per subunit from modification by 2, 3-butanedione. Unlike native enzyme, phenylglyoxal- and butanedione-modified enzyme samples are incapable of forming ternary complex. The results suggest that one arginyl residue per subunit participates in the functional binding of dUMP, presumably through electrostatic interaction with the 5'-phosphate moiety of the nucleotide. © 1979, American Chemical Society. All rights reserved.