Interaction of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase with Reactive Blue 2 and related dyes.
Steady-state kinetic analyses suggest that Pseudomonas ochraceae 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (4-carboxy-2-hydroxy-cis,cis-muconate-6-semialdehyde: NADP+ oxidoreductase [EC 1.2.1.45]) functions through an ordered BiBi mechanism. The enzyme binds one NADP+ molecule per subunit with a Kd of 4.8 +/- 0.8 microM. The enzyme is adsorbed to a Blue Sepharose CL-6B column and can be eluted therefrom with reagents having high affinity for the enzyme such as NADP+, NAD+, ATP, and Reactive Blue 2. Equilibrium dialysis and difference spectral titration show the binding of four molecules of Reactive Blue 2 per enzyme subunit. Two of these dye molecules show high-affinity binding with a Kd of 0.03 +/- 0.02 microM. The resulting 1: 2 enzyme-dye complex can be isolated by gel filtration on Bio-Gel P-6. The kinetic, spectroscopic, and chromatographic properties of the complex indicate that the dye-binding sites are different from the coenzyme binding site. The other two dye molecules, in contrast, bind loosely with a Kd of 0.8 +/- 0.5 microM to a site overlapping the coenzyme binding site. This is confirmed by the following findings: NADP+ effectively abolishes the difference spectrum associated with the enzyme-dye binding, and the slope of the double reciprocal plot showing the competitive inhibition of the dye (Ki = 0.20 +/- 0.02 microM) with respect to NADP+ linearly depends on the square of the dye concentration. Essentially similar results are also obtained with methoxy Reactive Blue 2 and Reactive Blue 4.(ABSTRACT TRUNCATED AT 250 WORDS)
Maruyama
K
K
Department of Chemistry, Faculty of General Education, Gifu University.
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004396:Coloring Agents / Q000378:metabolism* / Q000494:pharmacology; D003956:Dialysis; D007700:Kinetics; D011485:Protein Binding; D013053:Spectrophotometry; D014227:Triazines / Q000378:metabolism* / Q000494:pharmacology
D004396:Coloring Agents; D014227:Triazines; C012842:Cibacron Blue F 3GA; C021389:2-hydroxy-4-carboxymucconate-6-semialdehyde dehydrogenase; D000445:Aldehyde Oxidoreductases