Interaction of bacterial glucose-6-phosphate dehydrogenase with triazine dyes: a study by means of affinity partitioning and kinetic analysis.
The interaction of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Pseudomonas W6 with 33 triazine dyes bound to poly(ethylene glycol) has been screened by means of affinity partition in an aqueous two-phase system composed of poly-(ethylene glycol) and dextran. Regarding the strength of interaction expressed by the affinity partitioning effect three groups of dyes can be distinguished showing high, medium or low interaction to the enzyme. The different influence of the structurally related dyes Cibacronblue F3G-A, Procion Blue MX-3G and Procion Blue MX-R on the partitioning of glucose-6-phosphate dehydrogenase is discussed. Both coenzymes of this glucose-6-phosphate dehydrogenase, NADP+ and NAD+, are able to diminish the dye-enzyme interactions but to a different extent. In the case of Procion Red HE-3B less than 0.2 mM of NADP+ abolished completely the dye-protein interaction indicating that the dye binding site might be closely related to the nucleotide binding site. This does not hold for a series of other dyes revealing strong interactions to the enzyme. In these cases higher NADP(+)-concentrations are necessary to weaken the dye-enzyme interactions. Only small influences on the interaction between the dye and glucose-6-phosphate dehydrogenase were found with 1 mM NAD+. The different effects of the two coenzymes are discussed with respect to separate binding sites for them and to different conformational states induced. The competition of Procion Red HE-3B with NADP+ and NAD+ for the binding site of glucose-6-phosphate dehydrogenase was also demonstrated by inhibition studies. The Ki-values of Procion Red HE-3B with NAD+ and NADP+, respectively, were calculated.
Reuter
R
R
Institute of Biochemistry, Karl-Marx University, Leipzig, GDR.
Metz
P
P
Lorenz
G
G
Kopperschläger
G
G
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D004396:Coloring Agents / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D011092:Polyethylene Glycols; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014227:Triazines / Q000378:metabolism*
D001426:Bacterial Proteins; D004396:Coloring Agents; D014227:Triazines; D009243:NAD; D011092:Polyethylene Glycols; D005954:Glucosephosphate Dehydrogenase