J. Biochem, 1988, Vol. 103, No. 4 714-721
© 1988 Japanese Biochemical Society
research-article |
Interaction of 4-Carboxy-2-Hydroxymuconate-6-Semialdehyde Dehydrogenase with Reactive Blue 2 and Related Dyes
Department of Chemistry, Gifu University Gifu, Gifu 501-11
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 [EC] ]) functions through an ordered BiBi mechanism. The enzyme binds one NADP+ molecule per subunit with a Kd of 4.8±0.8 µM. 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 µM. 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 µM 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 (K1 =0.20±0.02 µM) 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. The binding of Blue Dextran-2000 to the enzyme is somewhat unlike that of Reactive Blue 2, possibly due to insufficient contact of the dye chromophore with the enzyme.