Evidence for an essential glutamyl residue in yeast hexokinase

Abstract

Yeast hexokinase is rapidly inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosyl ethyl ester. Sugar substrates afford 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 with their binding induces. It is concluded that a glutamyl residue is essential for yeast hexokinase activity and its catalytic function is discussed.