Evidence for an essential glutamyl residue in yeast hexokinase.

Yeast hexokinase is rapidly inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosyl ethyl ester. Sugar substrates afford a partial protection, which is increased by the addition of ADP. Inactivation of the enzyme takes place concomitantly with the incorporation of 1 mol of nitrotyrosine per mol of 50 000-dalton subunit. Exhaustive proteolytic digestion of the modified protein and isolation of the nitrotyrosyl peptide by affinity chromatography, followed by electrophoresis, lead to the identification of the modified residue as a glutamyl residue. This modification of hexokinase occurs without gross conformational changes. The enzyme still binds its substrates, though binding of the nucleotides is perturbed. While the substrates afford a partial protection, they increase the incorporation of nitrotyrosine ethyl ester into the enzyme. This may be attributed to local conformational changes which their binding induces. It is concluded that a glutamyl residue is essential for yeast hexokinase activity and its catalytic function is discussed.