J. Biol. Chem., Vol. 256, Issue 17, 8970-8976, Sep, 1981
Affinity labeling of chicken liver dihydrofolate reductase by a substituted 4,6-diaminodihydrotriazine bearing a terminal sulfonyl fluoride
AA Kumar, JH Mangum, DT Blankenship and JH Freisheim
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.
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