The structure of dihydrofolate reductase. I. Inactivation of bacterial dihydrofolate reductase concomitant with modification of a methionine residue at the active site.
Carboxymethylation by iodoacetate of dihydrofolate reductase from the amethopterin-resistant mutant Streptococcus faecium var. Durans strain A leads to a loss of enzymic activity. Amino acid analysis showed that methionine is the only amino acid residue significantly affected by iodoacetate under the experimental conditions, and this was confirmed by the use of [1-14-C]iodoacetate and ion exchange chromatography of the products obtained by acid hydrolysis of the modified enzyme. During loss of 90% of the activity a total of about 2 of the 7 methionine residues present in the enzymes are carboxymethylated. Over this range of activity loss the decrease is proportional to the number of methionine residues modified. Fluorescence-quenching experiments demonstrated that dissociation constants for complexes of inhibitors with the carboxymethylated enzyme were 20 to 30 times greater than dissociation constants for corresponding complexes with native enzyme. Similarly, equilibrium dialysis studies showed that dihydrofolate binding to the modified enzyme was decreased 10-fold compared with binding to the native enzyme. These data suggest that iodoacetate modifies one or more methionine residues at the binding site for dihydrofolate and inhibitors. In accordance with this view it was shown that enzyme can be protected from inactivation by the folate analogue aminopterin and to a lesser extent by folate and dehydrofolate. Enzyme carboxymethylated in the presence of aminopterin, and subsequently freed of the latter, was found to bind inhibitors and dihydrofolate as tightly as the native enzyme. It is concluded that the loss of enzyme activity is caused by carboxymethylation of at least 1 methionine residue which is at or near the binding site of dihydrofolate.
D001665:Binding Sites; D002942:Circular Dichroism; D003956:Dialysis; D004352:Drug Resistance, Microbial; D005493:Folic Acid Antagonists; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D008715:Methionine; D008727:Methotrexate / Q000494:pharmacology; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013291:Streptococcus / Q000187:drug effects / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase; D013997:Time Factors
D005493:Folic Acid Antagonists; D007461:Iodoacetates; D008715:Methionine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate