Affinity labeling of chicken liver dihydrofolate reductase by a substituted 4,6-diaminodihydrotriazine bearing a terminal sulfonyl fluoride.
Chicken liver dihydrofolate reductase is rapidly and stoichiometrically inactivated by a substituted 4,6-diaminodihydrotriazine containing a terminal benzenesulfonylfluoride (DTBSF). The substrate dihydrofolate largely prevents the enzyme inhibition by DTBSF, whereas NADPH had no effect, indicating that the inhibitor is bound at or near the folate site. Using radiolabeled inhibitor between 1.0 and 1.2 mol was incorporated/mol of enzyme (Mr = 21,651), following treatment with 8 M urea at 75 degrees C. Digestion of the maleylated, radiolabeled inhibitor-enzyme complex with trypsin and subsequent gel filtration on Sephadex G-50 SF yielded a single major peak of radioactivity. The covalently modified limited tryptic peptide was subsequently purified to homogeneity using high performance liquid chromatography. The radiolabeled tryptic peptide had the following sequence: Asn-Glu-Tyr (DTBS)-Lys-Tyr-Phe-Gln-Arg (residues 29-36). Automated Edman degradation of this peptide revealed that the radioactivity derived from the inhibitor was released at Step 3, identifying tyrosine-31 as the specific site of covalent attachment of the affinity label.
D000345:Affinity Labels / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002645:Chickens; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D013045:Species Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014227:Triazines / Q000494:pharmacology*
D000345:Affinity Labels; D000596:Amino Acids; D010446:Peptide Fragments; D014227:Triazines; C031776:NSC 127755; D013762:Tetrahydrofolate Dehydrogenase