Selective arylation of cysteine-237 of rabbit muscle aldolase with 4-chloro-7-nitrobenzofurazan.

At pH 7.0 and 25 degrees C, NBF-Cl (4-chloro-7-nitrobenzofurazan) reacts rapidly with rabbit muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) to yield a product with an absorption maximum at 402 nm, which is shifted to 422 nm upon acid denaturation. The reaction involves arylation of a single cysteine residue per subunit of tetrameric aldolase, as shown by the molar absorptivity of NBF-aldolase and by titration of sulfhydryl groups of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid), DTNB. The site of arylation appears to be Cys-237, which is the cysteine residue that reacts most rapidly with DTNB. The site of arylation appears to be Cys-237, which is the cysteine residue that reacts most rapidly with DTNB, and which is not essential for aldolase activity. Arylation of the enzyme is 13-20-times more rapid than that of model compounds. The relatively high rate of arylation is not due to medium effects, to an anomalously low pKa of Cys-237, or to the presence of a binding site for NBF-Cl, and is tentatively assigned to acid-base catalysis by other functional groups in the vicinity of the reactive sulfhydryl group. The NBF-Cl reaction provides the most efficient means of titrating Cys-237 residues in rabbit muscle aldolase.