The Extents of Formation of Cobalt(II)-Radical Intermediates in the Reactions with Different Substrates Catalysed by the Adenosylcobalamin-Dependent Enzyme Ethanolamine Ammonia-Lyase

Abstract

The reactions of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase [Clostridium sp.], with the good and relatively poor substrates 2-aminoethanol and (S)-2-aminopropanol, respectively, under conditions of saturation with substrate, were investigated by rapid freezing in conjunction with EPR spectroscopy and by stopped-flow spectrophotometry. In disagreement with earlier reports the reaction of 2-aminoethanol gave an EPR signal observed in rapid freezing experiments characteristic of a coupled Co(II)-free radical system. This signal was similar to, though not identical with, that obtained with (S)-2-aminopropanol. The steady-state level of the signal with 2-aminoethanol as substrate was 0.56 of that attained with (S)-2-aminopropanol. The results of these EPR experiments were consistent with stopped-flow data obtained under closely similar reaction conditions, the latter indicating a corresponding ratio of 0.64. The results also are consistent with those of a rapid wavelength scanning, stopped-flow spectrophotometric study.