Reaction of dihydrofolate reductase with dansyl chloride. Chemical modification of a sensitive lysine residue and fluorometric studies of the dansylated enzyme

Publisher Website
Google Scholar
Abstract
Dihydrofolate reductase [EC 1.5.1.3] from amethopterin-resistant Lactobacillus casei is virtually completely and irreversibly inactivated by relatively low concentrations of dansyl chloride. The complete inactivation can be correlated with the dansylation of a single lysine residue and .apprx. 90% quenching of protein fluorescence. This quenching phenomenon appears to be due partly to energy transfer from 1 or more excited state tryptophan residues to the covalently attached dansyl moiety. Under identical conditions lysine is not modified when the ternary complex of enzyme-NADPH-amethopterin is dansylated. The unreactive dansyl hydroxide protects the enzyme against dansyl chloride dependent inactivation, and fluorescence studies indicate a single ligand binding site (Kd = 1 .times. 10-4 M). The dimethylaminonaphthyl moiety of dansyl chloride may be directed to a hydrophobic region at or near the enzyme active center where a particularly susceptible lysine residue reacts to form a covalent bond with the reagent.