ASPARTATE-221 OF THYMIDYLATE SYNTHASE IS INVOLVED IN FOLATE COFACTOR BINDING AND IN CATALYSIS

Structural studies indicate that Asp 221 of Lactobacillus casei thymid ylate synthase forms a hydrogen bond network with the 2-amino and 3-im ino groups of the folate [Matthews, D, A. (1990) J. Mol. Biol. 214, 93 7-948; Finer-Moore, J, S. (1990) Biochemistry 29, 6977-6986] that has been proposed to participate in catalysis. We prepared a complete repl acement set of 19 mutants at position 221 of L. casei thymidylate synt hase. Of these, the only one with sufficient activity to complement gr owth of a thymidylate synthase-deficient host was the Cys mutant. To f urther elucidate the function of the Asp 221 side chain, seven thymidy late synthase 221 mutants were studied in detail with regard to cataly sis of dTMP formation and of thymidylate synthase partial reactions. M ost of the mutants bound the nucleotide substrate dUMP with only moder ate loss of binding affinity, indicating that the Asp side chain does not contribute to dUMP binding, Most of the mutants catalyzed the cofa ctor-independent dehalogenation of 5-bromodUMP; hence, the Asp side ch ain of TS is not essential for addition of the catalytic Cys residue t o the nucleotide substrate. Mutants showed decreased affinity for the folate cofactor, but those with side chains capable of hydrogen bond f ormation were less severely affected. Some of the mutants were capable of forming covalent thymidylate synthase-5-fluorodUMP - methylenetetr ahydrofolate complex; hence, the Asp side chain is not essential for s teps leading to the covalent complex. We conclude that the hydrogen bo nd network between Asp 221 and the folate cofactor contributes to cofa ctor binding and a catalytic step after formation of the covalent tern ary complex intermediate.