Modification of lysyl residues of dihydrofolate reductase with 2,4-pentanedione.

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) from an amethopterin-resistant strain of Lactobacillus casei was inactivated by 2,4-pentanedione. The inactivation appears to be due to the specific interaction of 2,4-pentanedione with lysyl residues. Inactivation is concomitant with with the modification of three lysyl residues. Both NADPH and dihydrofolate protect the enzyme against inactivation, suggesting that the critical residue(s) lies at or near their binding sites. Unlike native dihydrofolate reductase, 2,4-pentanedione-modified enzyme does not form binary complexes with either NADPH, dihydrofolate or amethopterin which are stable to gel filtration. Treatment of the modified enzyme with nucleophilic reagents such as hydroxylamine, failed to promote reactivation of the enzyme. Reactivation was achieved following gel filtration at pH 6.0 and was found to be dependent on the degree to which the enzyme was inactivated.