Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site.
Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005493:Folic Acid Antagonists; D005981:Glutathione Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D010453:Peptide Synthases / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis* / Q000302:isolation & purification
D005493:Folic Acid Antagonists; D012967:Sodium Dodecyl Sulfate; D013762:Tetrahydrofolate Dehydrogenase; D010453:Peptide Synthases; D005981:Glutathione Synthase; D008727:Methotrexate