Interaction of biodegradative threonine dehydratase (TD) of Escherichia coli with 8-azido-AMP, a photoaffinity analog of AMP

Stimulation of TD activity by AMP (Ka = 40 μM) is known to accompany the conversion of monomeric form of the enzyme to its tetramer and a decrease in the Km for threonine. In comparison, 8-azido-AMP (N3AMP) simulated TD activity only partially, lowered the Km for threonine and stabilized a dimeric form of the protein. Competition experiments revealed that N3AMP exerted an apparent dominant effect in counteracting the AMP-mediated enzyme activation, indicating complex mutual interactions between these ligands. UV irradiation of TD with increasing concentrations of 3H-N3AMP (up to 150 μM) resulted in gradual loss of enzyme activity and concomitant incorporation of N3AMP into protein; upon complete inactivation, 0.75 mol of N3AMP was bound per mol tetramer. The presence of AMP during photolysis reduced the extent of enzyme inactivation as well as the incorporation of N3AMP into protein. Photolabeling of TD with 20 μM 3H-N3AMP revealed one labeled tryptic peptide; at higher N3AMP concentrations (>300 μM), two labeled peptides were found, one of which was identical to that found with low N3AMP concentration. The cumulative results suggest that N3AMP can act as an allosteric modifier and that the peptide labeled at low N3AMP may represent the AMP binding site on the protein molecule