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) require proton transfers to and from O-4 and C-5 of the pyrimidine moiety of substrate dUMP. It has been proposed that one or more of three active 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 mutated to residues incapable of proton transfer, and the mutant enzymes were purified and tested for activity in the formation of dTMP and the dehalogenation of 5-bromo- and 5-iodo-dUMP. The dehalogenation reaction pathway shares at least two direct chemical counterparts with the TS reaction pathway which are believed to involve general acid/base catalysis-namely, the addition and elimination of the catalytic Cys of TS at C-6 of the pyrimidine substrate. Generally, the mutations had detrimental effects on dTMP synthesis with the triple mutant being completely inactive. In contrast, single mutants TS E601, and H199A and, interestingly, the triple mutant stripped of all three active site catalysts catalyzed the dehalogenation reaction as well as or better than the wild-type enzyme. It was concluded that addition and elimination reactions involving the 5.6-bond of pyrimidine substrates do not require general acid/base catalysis or, alternatively, the water molecules in the TS active site serve this role. The function(s) of the triad of general catalysts resides elsewhere in the reaction pathway leading to dTMP synthesis.
Huang
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Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448, USA.
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D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004735:Energy Transfer / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics; D016254:Mutagenesis, Insertional; D011522:Protons; D013938:Thymidine Monophosphate / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics
D011522:Protons; D013938:Thymidine Monophosphate; D013940:Thymidylate Synthase