Studies on Metapyrocatechase: IV. CIRCULAR DICHROISM AND OPTICAL ROTATORY DISPERSION

Abstract

Spectropolarimetric studies were performed to examine the conformation of metapyrocatechase in the absence and presence of the substrate, catechol. Metapyrocatechase, a ferrous ion containing dioxygenase, had an ordered structure with a relatively low content of α helix. This ordered structure was partially destroyed by treatment of the enzyme with urea or alkali, but not by resolution of the bound iron. Upon addition of catechol under anaerobic conditions, a new negative circular dichroic band appeared at 317 nm with a concomitant decrease at 300 nm, although the circular dichroic spectrum in the region below 250 nm was essentially unchanged. The circular dichroic band at 317 nm permitted a quantitative assessment of substrate binding and 3 moles of catechol were shown to be bound per mole of enzyme. Similar results were obtained when other enzymatically active substrate analogues were employed. On the other hand, the addition of nonmetabolizable competitive inhibitors did not alter the circular dichroic spectra of the enzyme itself significantly, although they reduced the magnitude of the circular dichroic band at 317 nm produced by the substrates. The magnitude of the circular dichroic band at 317 nm was stoichiometrically related to the amount of ferrous ion bound to the enzyme. Available evidence indicates that the observed asymmetricity induced by the substrate binding is due to a local conformational change around the substrate-binding site and the iron-binding site of the enzyme protein.