title: Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies.
abstract: When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002942:Circular Dichroism; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology*; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013388:Succinimides; D013438:Sulfhydryl Compounds / Q000032:analysis; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014364:Tryptophan / Q000032:analysis
chemical_list: D000596:Amino Acids; D013388:Succinimides; D013438:Sulfhydryl Compounds; D009249:NADP; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate