Isolation of a covalent steady-state intermediate in glutamate 60 mutants of thymidylate synthase.
Glutamate 60 of thymidylate synthase coordinates a hydrogen bond network important in proton transfer reactions to and from the substrate dUMP. The E60A and E60L mutants of Lactobacillus casei thymidylate synthase catalyzed tritium exchange from [5-3H]dUMP for solvent protons faster than dTMP formation, indicating accumulation of a steady-state intermediate and a change in partitioning of the intermediate. A covalent complex consisting of E60A or E60L thymidylate synthase, dUMP, and the cofactor CH2H4 folate was isolated on SDS-polyacrylamide gel electrophoresis and shown to be chemically and kinetically competent to form dTMP. These results provide proof of the formation of a covalent steady-state intermediate in the reaction pathway of thymidylate synthase and demonstrate that the rate-determining step in the mutants occurs during conversion of the covalent intermediate to dTMP.
Huang
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Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448.
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D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013763:Tetrahydrofolates / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry*
D017931:DNA Primers; D003856:Deoxyuracil Nucleotides; D005971:Glutamates; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase