The relation of photooxidized histidines in yeast enolase to enzymatic activity.

The photooxidation of yeast enolase using rose bengal (Westhead, 1965) has been reexamined. Four histidines per subunit are oxidized during complete inactivation but only one is critical for maintaining enzymatic activity. The bulk of the protection against photoinactivation is afforded by high concentrations of magnesium (midpoint concentration = 2.5 mm) but only in the presence of substrate, placing the critical histidine at or near the binding site of either catalytic or inhibitory magnesium. The tryptic peptide containing this histidine has been isolated by triethylaminoethyl cellulose chromatography, preparative-paper electrophoresis, and chromatography on phosphorylated cellulose. Amino acid analysis, aminoterminal determination, and electrophoretic migration give the structure: -His-(Asn, Leu)-Lys-. The peptide may be tentatively assigned as residues 180–183 in the enolase amino acid sequence (C. C. Q. Chin, F. Wold, and J. M. Brewer, Fed. Proc., 1978, 37, 1618). Binding of the chromophoric competitive inhibitor, 3-aminoenolpyruvate-2-phosphate, by photoinactivated enolase showed no changes in the dissociation constants or stoichiometry. However, the extinction coefficient at 295 nm of bound 3-aminoenolpyruvate-2-phosphate was reduced from 22,000 to 10,700 m^?1 cm^?1, indicating an alteration in the environment associated with the bound inhibitor. The critical histidine does not appear to be necessary for substrate-inhibitor binding but is required for enzymatic activity.