The inactivation of yeast enolase by 2,3-butanedione

Yeast enolase has been reacted with 2,3-butanedione in borate buffer and the number of arginine residues modified has been measured as a function of enzymatic activity. Activity losses are linear with respect to arginine modification, with complete inactivation being correlated with the alteration of one arginine per subunit. The tryptic peptide whose arginine is modified has been isolated using O-(triethylaminoethyl)-cellulose chromatography and paper electrophoresis and is a dipeptide, leucyl-arginine. Protection against loss of activity is afforded by the addition of substrate, 2-phosphoglyceric acid, or a competitive inhibitor, 3-aminoenolpyruvate-2-phosphate (AEP), and Mg; elimination of the Mg, a prerequisite for substrate or inhibitor binding, eliminates the protection afforded by substrate. It is suggested that a single arginine residue per subunit of enolase is necessary for enzymatic activity and is located at or near the substrate binding site (active site). The binding of a chromophoric competitive inhibitor, AEP, to native and butanedione-reacted enolase was examined by spectrophotometric titrations. Compared to the native enzyme, the butanedione-inactivated enolase binds AEP, but with an extinction coefficient for the inhibitor that is about two-thirds of the native value; the binding affinities are comparable. The strong binding of the modified enzyme for AEP suggests that the arginine, although critical for enzymatic activity, is not significantly involved in the overall binding of the inhibitor.