Catalytic cysteine of thymidylate synthase is activated upon substrate binding

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in cataly sis has been characterized by kinetic and crystallographic studies. Positio n 167 variants including S167A, S167N S167D, S167C, S167G, S167L, S167T, an d S167V were generated by site-directed mutagenesis. Only S167A, S167G, S16 7T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that muta nt enzymes exhibited kcat values 1.1-95-fold lower than that of the wild-ty pe enzyme. Relative to wild-type TS, K-m values of the mutant enzymes for 2 '-deoxyuridylate (dUMP) were 5-90 times higher, while K-m values for 5,10-m ethylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rat e of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstr ate, by mutant TSs was analyzed and showed that only S167A and S167G cataly zed the dehalogenation reaction and values of k(cat)/K-m for the mutant enz ymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-ste ady state kinetics of ternary complex formation revealed that the productiv e binding of CH2H4-folate is weaker to mutant TSs than to the wild-type enz yme. Chemical transformation constants (k(chem)) for the mutant enzymes wer e lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to eithe r 1.7 Angstrom (S167A and S167T) or 2.6 degrees (S167C). The high-resolutio n data sets were refined to a R-crys of 19.9%. In the crystals some cystein e residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyet hyl)thiocysteine. The pattern of derivatization indicates that in the absen ce of bound substrate the catalytic cysteine is not more reactive than othe r cysteines. It is proposed that the catalytic cysteine is activated by sub strate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the tran sfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.