ELECTROSTATIC GUIDANCE OF CATALYSIS BY A CONSERVED GLUTAMIC-ACID IN ESCHERICHIA-COLI DTMP SYNTHASE AND BACTERIOPHAGE-T4 DCMP HYDROXYMETHYLASE

Thymidylate synthase (TS) and dCMP hydroxymethylase (CH) are homologou s enzymes which catalyze the alkylation of C5 of pyrimidine nucleotide s, One of the first catalytic steps is isomerization of the alkyl dono r, methylenetetrahydrofolate, from its N5,N10 bridged form to the N5 i minium ion upon enzyme binding. Glu58 in TS has been postulated [Matth ews et al. (1990) J. Mol. Biol. 214, 937-948] to be involved in this i somerization and the deprotonation of C5 of the nucleotide. Substituti on by Asp or Gin of Glu58 in Escherichia coil TS, or of the correspond ing Glu60 in CH from phage T4, decreases the activity of either enzyme . Alkylation is slowed much more than deprotonation, indicating uncoup ling of steps which are tightly coupled for the wild-type enzymes. The data support minor roles for Glu58/60 in nucleotide binding and in is omerization of methylenetetrahydrofolate, but no major roles in nucleo tide deprotonation, product dissociation, or hydration catalyzed by CH . The primary role of Glu58/60 is to accelerate bond cleavage between N5 of tetrahydrofolate and the methylene being transferred, The influe nce of Glu58/60 on the rate of bond cleavage is proposed to arise from electrostatic destabilization, due to the proximity of the glutamyl c arboxylate, of the anionic species formed when C5 of the nucleotide is deprotonated. The proposal explains the uncoupling of deprotonation a nd alkylation with the Glu58/60 variants and the reduced kinetic isoto pe effect on hydride transfer for TS(Glu58Gln). The inability of 5-dea zatetrahydrofolate to stimulate enzyme-catalyzed tritium exchange from [5-H-3]nucleotides into solvent suggests that N5 of tetrahydrofolate is the base which deprotonates the nucleotide.