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

Abstract

1. A number of reactive triazine dyes specifically and irreversibly inactive yeast hexokinase at pH 8.5 and 33 degrees C. Under these conditions, the enzyme is readily inactivated by 100 microM-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. 2. 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. 3. Quantitatively inhibited hexokinase contains approx. 1 mol of dye per mol of monomer of mol.wt. 51000. The inhibition is irreversible and activity cannot be recovered on incubation with high concentration (20 mM) of ATP or D-glucose. 4. 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 microM to 41.6 microM. Binding of the dye to hexokinase is accompanied by characteristic spectral changes in the range 560-700 nm. 5. Mg2+ 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. These effects can be exploited to purify hexokinase from crude yeast extracts. 6. The specific active-site-directed binding of triazine dyes to yeast hexokinase is interpreted in terms of the crystallographic structure of the hexokinase monomer.