The interaction of yeast hexokinase with Procion Green H-4G

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Abstract
A number of reactive triazine dyes specifically and irreversibly inactivate yeast hexokinase at pH 8.5 and 33.degree. C. Under these conditions, the enzyme is readily inactivated by 100 .mu.M-Procion Green H-4G, Blue H-B, Turquoise H-7G and Turquoise H-A, is less readily inactivated by Procion Brown H-2G, Green HE-4BD, Red HE-3B and Yellow H-5G and is not inactivated at all by Procion Yellow H-A. The inactivation of hexokinase by Procion Green H-4G is competitively inhibited by the adenine nucleotides ATP and ADP and the sugar substrates D-glucose, D-mannose and D-fructose but not by nonsubstrates such as D-arabinose and D-galactose. Quantitatively inhibited hexokinase contains .apprx. 1 mol of dye/mol of monomer of MW 51,000. The inhibition is irreversible and activity cannot be recovered on incubation with high concentrations (20 mM) of ATP or D-glucose. Mg2+ protects the enzyme against inactivation by Procion Green H-4G but enhances the rate of inactivation by all the other Procion dyes tested. In the presence of 10 mM-Mg2+ the apparent dissociation constant between enzyme and dye is reduced from 199.0 .mu.M-41.6 .mu.M. Binding of the dye to hexokinase is accompanied by characteristic spectral changes in the range 560-700 nm. Mg1+ promotes binding of yeast hexokinase to agarose-immobilized Procion Green H-4G but not to the other dyes tested. Elution could be effected by omission of Mg2+ from the column irrigants or by inclusion of MgATP or D-glucose but not by D-galactose. The specific active-site-directed binding of triazine dyes to yeast hexokinase is interpreted in terms of the crystallographic structure of the hexokinase monomer.

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