Ribonucleoside diphosphate reductase: Formation of active and inactive complexes of proteins B1 and B2

Abstract

Ribonucleoside diphosphate reductase from Escherichia coli B separates during purification into two catalytically inactive subunits, proteins B1 and B2. When the subunits are mixed, enzyme activity is readily regenerated. Sucrose gradient centrifugation indicated that protein B1 (sedimentation coefficient 7.8 s) and protein B2 (5.5 s) combined in the presence of magnesium ions to form a catalytically active complex. Under conditions which minimized dissociation this complex had a sedimentation coefficient of 9.7 s and consisted of equimolar amounts of the two subunits. The activity and substrate-specificity of the enzyme are modulated by nucleoside triphosphates which act as allosteric effectors. Stimulatory effectors, such as ATP and dTTP, did not appreciably change the sedimentation coefficient of the complex. However, in the presence of the negative effector dATP, at concentrations which inhibit enzyme activity, the 9.7 s complex was replaced by a heavy species with a sedimentation coefficient of 15.5 s. This heavy complex contained proteins B1 and B2 in equimolar amounts. A similar heavy complex was also formed with mixtures of other nucleoside triphosphates which inhibit the enzyme. On the other hand, the formation of the heavy complex was prevented by ATP at concentrations which reverse inhibition by dATP. We conclude from our results: (1) that the 9.7 s complex represents the active form of ribonucleoside diphosphate reductase; (2) that the 15.5 s complex represents an inactive form of the enzyme, and (3) that both complexes contain equimolar amounts of each subunit. From consideration of the differences in sedimentation coefficients we propose that the 15.5 s complex is a dimer of the 9.7 s complex.