Dihydrofolate reductase from chicken liver contains four tryptophan residues per mole (mol wt 22,000), as determined by: (a) the absorbancy ratio at 280 and 288 mμ at pH 6.5 in the presence of 6 m guanidinium chloride; or (b) the decrease in absorbancy at 278 mμ after treatment of the enzyme with excess N-bromosuccinimide at pH 4.0 in the presence of 5.3 m urea. Between pH 6 and 8, and in the absence of urea, the action of W-bromosuccinimide on the enzyme occurs in two stages. Relatively low levels of N-bromosuccinimide (up to 4 moles/mole of enzyme) cause a two- to threefold increase in catalytic activity without any appreciable destruction of the tryptophans. This activation is probably due to oxidation of a sulfhydryl group on the protein since it can be reversed by dithiothreitol. Higher concentrations of N-bromosuccinimide (ca. 10 moles/mole of enzyme) result in tryptophan oxidation with a concomitant decrease in enzymatic activity. Complete loss of activity, however, corresponds to the oxidation of only one tryptophan residue. Incubation of the enzyme with either reduced triphosphopyridine nucleotide or dihydrofolate affords protection against the effects of N-bromosuccinimide. These results indicate that at least one tryptophan residue, and a sulfhydryl group as well, are located at or near the substrate binding sites of the enzyme, and that oxidation of these moieties by N-bromosuccinimide leads to conformational changes in the protein, either at the active site or at some other location which, in turn, can affect the active site. © 1969, American Chemical Society. All rights reserved.
