Rabbit muscle enolase also has essential arginyl residues

Recent studies have attempted to clarify the roles of various amino acid residues in the catalytic activity of yeast enolase. The yeast enzyme has a single essential arginyl residue per subunit [l-3] which is likely involved’in binding the phosphate moiety of substrates to the active site [Z]. In addition, the enzyme can be inactivated by the modification of carboxyl [4,5] and histidyl [G-8] residues at the active site, but the roles played by these amino acid residues is less well defined. Inactivation of yeast enolase has also been correlated with the modification of me~ionyl residues [9,10] and cysteinyl residues under denaturing conditions [ 111, but in neither case are the modified residues thought to be involved in the catalytic mechanism. Similarly, lysyl residues are not involved in substrate binding or catalysis [8]. Although there have been comparative studies on the structural and catalytic properties of yeast and rabbit muscle enolases [ 10,121, little information is available on catalytically functional amino acid residues in the muscle enzyme. It has been shown that glycidol phosphate, a substrate analogue, inactivates rabbit muscle enolase by modification of a carboxyl group at the active site [ 13 ,141, while cysteinyl residues have been shown to be non-essential [ 15 ,161. Here we report that rabbit muscle enolase, like the yeast enzyme [l-3], has a single essential arginyl residue per subunit which likely serves to bind the anionic substrates to the active site. This conclusion is based on chemical modification with butanedione in borate buffer, a system known to be highly selective for the modification of arginyl residues in proteins [ 1,2,17-201.