ACTIVE-SITE GENERAL CATALYSTS ARE NOT NECESSARY FOR SOME PROTON-TRANSFER REACTIONS OF THYMIDYLATE SYNTHASE

Several steps of the reaction catalyzed by thymidylate synthase (TS) r equire proton transfers to and from O-4 and C-5 of the pyrimidine moie ty of substrate dUMP. It has been proposed that one or more of three a ctive site residues--Glu60, His199, and Asn229--together with ordered water molecules serve as general catalysts in facilitating such proton transfers. These three residues, individually and together, were muta ted to residues incapable of proton transfer, and the mutant enzymes w ere purified and tested for activity in the formation of dTMP and the dehalogenation of 5-bromo- and 5-iodo-dUMP. The dehalogenation reactio n pathway shares at least two direct chemical counterparts with the TS reaction pathway which are believed to involve general acid/base cata lysis--namely, the addition and elimination of the catalytic Cys of TS at C-6 of the pyrimidine substrate. Generally, the mutations had detr imental effects on dTMP synthesis with the triple mutant being complet ely inactive. In contrast, single mutants TS E60L and H199A and, inter estingly, the triple mutant stripped of all three active site catalyst s catalyzed the dehalogenation reaction as well as or better than the wild-type enzyme. It was concluded that addition and elimination react ions involving the 5,6-bond of pyrimidine substrates do not require ge neral acid/base catalysis or, alternatively, the water molecules in th e TS active site serve this role. The function(s) of the triad of gene ral catalysts resides elsewhere in the reaction pathway leading to dTM P synthesis.