Studies on pyrocatechase from a soil bacterium

Abstract

A microorganism belonging to $\mbox{\underline{Pseudomonadaceae}}$ was screened from soil. The pyrocatechase was partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration. The final yield was 12\% with the specific activity of 5 u mole per min per mg-protein. The results of the general characterization were in general in accord with those from other isofunctional enzymes. It was active between pH 7 and pH 10. The optimum temperature was 35$^\circ$C. The Arrhenius activation energy was 12.6 kcal per mole. The enzyme was stablest at pH 9. But it lost part of its activity rapidly for the initial 30 hours resulting in 60\% of residual activity in 10 days. The molecular weight was found to be less than $7\times10^4$ by Sephadex G-75 chromatography. The Michaelis parameter, Km was 1.9 uM for catechol. The Lineweaver-Burk plot showed noncompetitive pattern when the oxygen concentration was varied below saturation. The enzyme was seriously inhibited by heavy metals and sulfhydryl agents. PCMB showed strong inhibition immediately while DTNB did only after incubation for 12 hours. The activity was partially restored by the addition of glutathione. The metal chelating agents for divalent ions such as o-phenanthroline or a,$a``$ -dipyridyl were ineffective together with that for trivalent ion, Tiron. By studying the effect of pH on the kinetic parameters, and from the experiment of photooxidation it was supposed that the imidazole group may participate in the catalysis. The evolution of the gene for pyrocatechase was discussed on the basis of the comparison of the isofunctional enzymes from diverse microorganisms. Finally, in addition to the presently known mechanism, a more concrete and probable mechanism involving the imidazole group as a catalytic group at the enzyme active site was proposed. The imidazole group forms a hydrogen bond with hydroxyl oxygen of catechol. A partial positive charge is developped on the carbon atom b...