Chemical Studies on Yeast Hexokinase. Specific Modification of a Single Tyrosyl Residue with 1‐Ethyl‐3‐(3‐Dimethylaminopropyl) Carbodiimide

Abstract

Of the 15 tyrosyl residues/subunit of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase EC 2.7.1.1) only 1 residue is specifically modified at pH 8.0 and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. The acylation of this single tyrosyl residue leads to the loss of the enzyme activities (hexokinase and ATPase) by a 1st-order process, which is fully reversed by treatment with hydroxylamine. ATP does not protect the enzyme against chemical modification and inactivation; however, glucose exerts a noticeable though indirect protection effect against chemical modification and inactivation. The chemically modified enzyme, purified by column chromatography, has 14% of the activity of the native enzyme, but the Km for ATP.cntdot.Mg or glucose remains unchanged as does the pH optimum of activity. Results of conformational studies (ultracentrifugation, fluorescence, thermostability and chemical reactivity of the sulfhydryl groups) indicate that the decrease of enzyme activity due to the modification of the tyrosyl residue is related to a localized perturbation of the enzyme active-center region.