The photoreactive AMP analog, g-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the K , for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 pM and 1.5 pM, respectively, and the maximum stimulation by 8-azido-AMP was 25% of that seen with AMP. Gel-filtration experiments revealed that 8-azido-AMP stabilized a dimeric form of the enzyme, whereas AMP promoted a tetrameric species. When present together, AMP and 8-azido-AMP showed mutual competition in influencing catalytic activity as well as the conformational state of the protein. Since the initial report [ 13 that the biodegradative threonine dehydratase of Escherichia coli, which catalyzes the pyridoxal phosphate-dependent dehydration of threonine and serine to ammonia and corresponding 2-0x0 acids, is stimulated by AMP, considerable evidence has been gathered on the effect of this nucleotide on the conformational states, catalytic mechanism, and regulation of enzyme activity by various cellular metabolites. For example, the enzyme can bind 4 mol AMP/mol protein [2], and the AMP-free enzyme exists as a protomer of M , 35000, whereas the AMP-bound form is a tetramer of M , 140000 which exhibits a 25-fold decrease in the K , for threonine [2-41. The stimulatory effect of AMP has been correlated with obligatory oligomerization of the protomeric species [4]. Furthermore, allosteric inhibition of enzyme activity [5 -71 and catabolite inactivation by covalent protein modification of the dehydratase by intermediary metabolites [7 -91 are known to be precisely regulated by subtle variations in the concentration of AMP. Nevertheless, the binding site for AMP on the enzyme molecule remains unidentified. Recently, the complete amino acid sequence of the E. coli threonine dehydratase has been determined in this laboratory from the nucleotide sequence of the cloned gene [lo, 111, and the active-site pyridoxal-phosphate-bound lysine residue involved in the dehydration reaction has been localized on the protein primary structure [ I l , 121. This report is concerned with photolabeling of the binding site of 8-azido-AMP, a photoaffinity analog of AMP. The experiments revealed that 8-azido-AMP can mimic the allosteric modifier AMP and binds to a unique site on the dehydratase molecule which most likely represents the binding region of AMP.