Interaction of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase with Reactive Blue 2 and related dyes.

Abstract:

: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)

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Maruyama K

doi

10.1093/oxfordjournals.jbchem.a122334

subject

Has Abstract

pub_date

1988-04-01 00:00:00

pages

714-21

issue

4

eissn

0021-924X

issn

1756-2651

journal_volume

103

pub_type

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