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Metal-ion-promoted binding of triazine dyes to proteins. The interaction of Cibacron Blue F3G-A with yeast hexokinase.

ABSTRACT: Bivalent metal ions, particularly Zn2+ and other members of the first-row transition series, promote irreversible inactivation of yeast hexokinase by Cibacron Blue F3G-A at a site competitive with both ATP and D-glucose. Difference spectroscopy indicates that the protein-dye dissociation constant is decreased from 250 micrometers in the absence of metal ions to less than 100 micrometers in the presence of appropriate concentrations of metal ions, with specificity displayed in the sequence of Zn2+ greater than Cu2+ greater than Ni2+ greater than Mn2+. Quantitative inactivation of yeast hexokinase leads to the incorporation of approx. 1 mol of Cibacron Blue F3G-A/mol of subunit of mol. wt. 51 000 in both the presence and the absence of metal ion. These results suggest the formation of a highly specific ternary complex involving enzyme, dye and metal ion at the active-site region of the enzyme, and correlate well with the known effects of metal ions in promoting the binding of hexokinase to immobilized Cibacron Blue F3G-A.

SUBMITTER: Hughes P

PROVIDER: S-EPMC1158501 | biostudies-other | 1982 Aug

REPOSITORIES: biostudies-other

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Project description:Yeast hexokinase, a homodimer (100 kDa), is an important enzyme in the glycolytic pathway. Although Cibacron Blue 3G-A (Reactive Blue 2) has been previously shown to inactivate yeast hexokinase, no comprehensive study exists concerning the nature of interaction(s) between hexokinase and the blue dye. A comparison of the computer-generated three-dimensional (3D) representations showed considerable overlap of the purine ring of ATP, a nucleotide substrate of hexokinase, with the hydrophobic anthraquinone moiety of the blue dye. The visible spectrum of the blue dye showed a characteristic absorption band centred at 628 nm. The visible difference spectrum of increasing concentration of the dye and the same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum and an isobestic point at 683, 585, and 655 nm respectively. The visible difference spectrum of the blue dye and the dye in 50% ethylene glycol showed a maximum and a minimum at 660 and 570 nm respectively. The visible difference spectrum of the blue dye in the presence of the dye and hexokinase modified at the active site by pyridoxal phosphate, iodoacetamide and o-phthalaldehyde was devoid of bands characteristic of the hexokinase-blue dye complex. Size-exclusion-chromatographic studies in the absence or presence of guanidinium chloride showed that the enzyme inactivated by the blue dye was co-eluted with the unmodified enzyme. The dialysis residue obtained after extensive dialysis of the gel-filtered complex, against a buffer of high ionic strength, showed an absorption maximum at 655 nm characteristic of the dye-enzyme complex. Inactivation data when analysed by 'Kitz-Wilson'-type kinetics for an irreversible inhibitor, yielded values of 0.05 min-1 and 92 microM for maximum rate of inactivation (k3) and dissociation constant (Kd) for the enzyme-dye complex respectively. Sugar and nucleotide substrates protected hexokinase against inactivation by the blue dye. About 2 mol of the blue dye bound per mol of hexokinase after complete inactivation. The inactivated enzyme could not be re-activated in the presence of 1 M NaCl. These results suggest that Cibacron Blue 3G-A inactivated hexokinase by an irreversible adduct formation at or near the active-site. Spectral and kinetic studies coupled with an analysis of the 3D representations of model compounds corresponding to the substructures of the blue dye suggest that 1-amino-4-(N-phenylamino)anthraquinone-2-sulphonic acid part of the blue dye may represent the minimum structure of Cibacron Blue 3G-A necessary to bind hexokinase.(ABSTRACT TRUNCATED AT 400 WORDS)

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Metal-ion-promoted binding of triazine dyes to proteins. The interaction of Cibacron Blue F3G-A with yeast hexokinase.

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