Studies on Pyrocatechase

Abstract

Pyrocatechasewas purified from extracts of benzoateinduced cells of Pseudomonas arvilta.The most purified preparation had a specific activity of 29.6, about 50% higher than that previously reported, and was homogeneous as judged by ultracentrifugation and by electrophoresis.The molecular weight was estimated to be approximately 90,000.The enzyme contained 2 g atoms of iron per mole of enzyme protein.Although the chemical determination of the valency state of iron has given inconclusive results, experiments with various chelating agents and the effects of oxidizing and reducing agents indicated that the iron in the native enzyme may be in the trivalent state.The concentrated solution of highly purified pyrocatechase had a pronounced red color with a broad absorption between 390 and 650 mp.The peak was at about 440 ml.c, and the molecular absorbance at 440 rnp was estimated to be 46'70.The trivalent iron bound to the enzyme appears to be responsible for the visible absorption band and functions as an integral part of the enzyme, since under a variety of conditions a loss of enzyme activity occurred in parallel with the disappearance of the absorbance in the visible range.By the addition of the substrate, catechol, under anaerobic conditions, the color of the enzyme solution changed to greyish blue with a concurrent increase of the absorbance