Specific acylation of the histidine residues of yeast hexokinase with diethylpyrocarbonate at pH 6.1 or 7.5 leads to a partial and reversible inactivation of the enzyme.The carboethoxylation of the histidine residues at pH 7.5 was faster than at 6.1 and the loss in activity was also higher.However the difference in the amount of inactivation was related to a greater instability of the carboethoxylated enzyme at pH 7.5.When the carboethoxylation was carried out at pH 7.5 and at 0 "C, only the histidines residues were modified (nine residues per enzyme subunit) and still 4001, of the activity was left.D-Glucose protects the enzyme activity in the course of the carboethoxylation at pH 7.5, but has no effect a t pH 6.1. However the rate and number of histidines acylated were the same as when the experiments were carried in absence of the substrate.All these results clearly indicate that the histidine residues are not implicated either in the catalytic or in the substrate binding site of yeast hexokinase.This was also confirmed by showing that the 600/, inactivated enzyme had the same K , as the native enzyme for both substrates D-glucose and Mg * ATP. Only v was affected.The inactivation effect of the carboethoxylation was not related to a displacement of the pH optimum of the activity, since the enzymes species having five and eight histidine residues modifiedrespectively in absence and presence of D-glucose at pH 7.5 gave the same pH profile of activity as the native enzyme.Absorption spectroscopy, fluorescence and ultracentrifugation studies have shown that the loss of enzymic activity in the course of the modification of the histidine residues is only related to changes of the protein conformation.Numerous investigations have been carried out on the mode of action of yeast hexokinase. I n contrast, the nature of the amino acid residues participating in the catalytic process are still unknown.I n a previous work [l], we have observed that yeast hexokinase is inactivated by photooxidation in presence of methylene blue. In the course ofthe photodynamic process, the loss of enzyme activity occurs by a biphasic process. At the initial rate of photooxidation one histidyl residues is modified with concommitant loss of 50 of the activity and change of the tertiary structure of the protein. I n the second phase of the photooxidation, multiple chemical effects were observed (oxidation of another 5 histidyl, 2 cysteinyl, 2 tryptophyl and 10 methionyl residues) which rendered the interpretation of the mechanism of photoinactivation more hazardous. Eur. J. Biochem. 39 (1973) Histidine groups have been described to be involved in the catalysis of several phosphoryl-transfer enzymes [2-41. From the photooxidation studies of hexokinase, we were unable to include or exclude definitively the role of this residue in the mechanism of action of the enzyme.Diethylpyrocarbonate has been shown, by several authors, to be a specific acylating agent of the histidine residues in proteins [4-91.I n this report the effect of this reagent ...