Activation and oligomerization of immobilized biodegradative L-threonine dehydrase

Biodegradative L-threonine dehydrase of Escherichia coli (radio labeled with [3H]pyridoxine) was immobilized on CNBr-activated Cl-Sepharose. The specific catalytic activity and S0.5 values of the matrix-bound dehydrase in the presence of AMP were similar to those of the soluble oligomeric enzyme in the presence of AMP (matrix-bound, associated dehydrase). When the bound dehydrase was washed with AMP-free buffer, about 50% of the bound dehydrase was removed and about 50% remained attached, as measured by radioactivity. The resulting matrix-bound, dissociated dehydrase possessed activity in the absence of AMP as is characteristic of soluble, unactivated dehydrase. The bound, dissociated dehydrase was capable of binding nearly an equal amount of soluble dehydrase in the presence of AMP; this treatment raised the specific enzyme activity of the bound dehydrase to 83% of that of the original matrix-bound, associated dehydrase. These observations correlate with the effects of AMP on the activity and quaternary structure of soluble dehydrase. When AMP was added to the matrix-bound, dissociated dehydrase, the activation observed was only a small fraction of that obtained with soluble dehydrase plus AMP. The failure of AMP to activate the major fraction of immobilized dehydrase monomer strongly suggests that dimerization is required in the activation by AMP.