Interaction of bis(1-anilino-8-naphthalenesulfonate) with yeast hexokinase: a steady-state fluorescence study.
Bis(1-analino-8-naphthalenesulfonate) (bis-ANS) is a useful probe for hydrophobic areas on protein molecules and it has been proposed that it has a general affinity for the nucleotide binding site(s). There appear to be two different classes of binding sites for bis-ANS on hexokinase and these can be tentatively assigned as primary and secondary binding sites. The rate of binding of bis-ANS at the primary binding site is fast, whereas binding at secondary site(s) is slow. The slow increase in the fluorescence intensity on binding with bis-ANS is not due to conformational change in the enzyme, which may lead to the increase in the quantum yield of the bound dye. Circular dichroism measurements indicate that there is no significant change in the secondary structure on binding with this probe. In the presence of saturating amounts of glucose, the increase in fluorescence intensity due to binding at the secondary binding site(s) is significantly lowered. This indicates that glucose-induced conformational change has been sensed by this probe. From kinetic studies, it has been observed that bis-ANS is an effective competitive inhibitor of yeast hexokinase with respect to ATP. The stoichiometry of binding of this fluorescent probe is about one per subunit at the primary site both in the presence and absence of glucose, and the dissociation constant of bis-ANS is unaffected by glucose. It is possible to decrease significantly the amount of fluorescence intensity at the primary site by nucleotides. These results indicate that bis-ANS interacts at the site where nucleotide interacts. Energy transfer experiments indicate the proximity of some tryptophan(s) and bound bis-ANS molecule(s).
Maity
H
H
Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India. maity@sunset.backbone.olemiss.edu
Kasturi
S R
SR
D000244:Adenosine Diphosphate / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism*; D014364:Tryptophan; D015003:Yeasts / Q000201:enzymology
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D000244:Adenosine Diphosphate; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D006593:Hexokinase; D005947:Glucose