Catalytic role of histidine 147 in Escherichia coli thymidylate synthase.
Nine mutant thymidylate synthases were isolated that only differed in sequence at position 147. The wild-type enzyme (which had a histidine residue at 147) and mutant enzymes were purified to near homogeneity and their kinetic properties were compared. Although the kcat values for the mutant enzymes were 10-10,000-fold lower than for the wild-type enzyme, the Km values for both 2'-deoxyuridylate and 5,10-methylenetetrahydrofolate were nearly identical for all the enzymes indicating that His-147 is not significantly involved in initial substrate binding. By comparing the wild-type (His-147) to the glycine (Gly-147) enzyme, the side chain of His-147 was estimated to lower the activation energy of the catalytic step by 1.6-2.9 kcal mol-1. In contrast to the wild-type enzyme, the activity of the Gly-147 enzyme decreased when the pH was raised above 7.5. The activity loss coincided with the deprotonation of a residue that had a pKa of 9.46 +/- 0.2 and an enthalpy of ionization (delta Hion) of 12.1 +/- 0.9. These values are consistent with the involvement of a lysine or an arginine residue in the catalytic process. An inspection of the rates of ternary complex formation among enzyme, 5-fluoro-2'-deoxyuridylate, and 5,10-methylenetetrahydrofolate for the mutant enzymes indicated that His-147 is not needed for the proton removal from C-5 of 2'-deoxyuridylate but rather participates in an initial catalytic step and alters the pKa value of a catalytically important lysine or arginine residue.
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Division of Molecular Genetics and Microbiology, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709.
Yates
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BB
Atashi
J
J
Dallas
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WS
D000595:Amino Acid Sequence; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000302:isolation & purification; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin
D003062:Codon; D010446:Peptide Fragments; D006639:Histidine; D013940:Thymidylate Synthase; D014357:Trypsin