Interaction between ethanolamine ammonia-lyase and methylcobalamin. Half-site reactivity with an adenosylcobalamin-dependent enzyme

Abstract

The adenosylcobalamin-dependent enzyme ethanolamine ammonia-lyase contains two active sites per molecule. The effects of methylcobalamin on the properties of this enzyme differ qualitatively depending on whether one or both of these sites is occupied by the cobamide. At 0.5 mol of methylcobalamin/mol of active sites, catalytic activity fell rapidly to approximately 30% of control levels, thereafter remaining constant for an hour. With the partially inhibited enzyme, Km values for ethanolamine and adenosylcobalamin were 5.5 muM and 1.6 muM, respectively, values that do not differ significantly from those of uninhibited enzyme. When the methylcobalamin per active site ratio was increased to 1, the decline in activity became progressive with time, eventually falling to levels much lower than seen at a cobamide per active site ratio of 0.5. Methylcobalamin also promotes the formation of a complex stable to gel filtration between ethanolamine and enzyme. Complex formation increased with increasing methylcobalamin per active site ratios up to a ratio of 0.7/1, at which point 0.5 mol of ethanol/mol of active sites was taken up. Ethanolamine uptake did not increase at higher methylcobalamin to active sites ratios. Methylcobalamin itself was taken up by enzyme, forming a complex containing 0.5 mol of methylcobalamin/mol of active sites that was stable to gel filtration. Measurement by the technique of Hummel and Dreyer ((1962) Biochim. Biophys. Acta 63, 530-532), however, showed one methylcobalamin binding site per active site. The formation of enzyme-ligand complexes stable to gel filtration was not affected by 5'-deoxyadenosine nor did 5'-deoxyadenosine by itself promote the formation of a stable complex between enzyme and ethanolamine. These observations were interpreted as evidence indicating half-site reactivity of ethanolamine ammonia-lyase with methylcobalamin. Comparison with previous results suggested that this half-site reactivity was an epiphenomenon not related to catalysis.