Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8-azido-AMP.
The photoreactive AMP analog, 8-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the Km for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 microM and 1.5 microM, 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. Photolabeling of AMP-free dehydratase with 8-azido-[2-3H]AMP resulted in a time and concentration-dependent enzyme inactivation and concomitant incorporation of 8-azido-AMP into protein. At low 8-azido-AMP concentrations, incorporation of about 1 mol 8-azido-AMP/mol dehydratase tetramer was correlated with almost complete inactivation of the enzyme. The presence of AMP in the photolabeling reaction greatly reduced the extent of enzyme inactivation and 8-azido-AMP binding. Ultraviolet irradiation with 20 microM 3H-labeled 8-azido-AMP revealed one tryptic peptide, Thr230-Thr-Gly-Thr-Leu-Ala-Asp-Gly-Cys-Asp-Val-Ser-Arg242, with bound radioactivity. This peptide, labeled at low concentration of 8-azido-AMP, most likely represents the AMP-binding region on the dehydratase molecule.
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Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
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D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000495:Allosteric Site; D001386:Azides / Q000378:metabolism*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D013913:Threonine Dehydratase / Q000378:metabolism*; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D000249:Adenosine Monophosphate; C021291:8-azidoadenosine 5'-monophosphate; D014357:Trypsin; D013913:Threonine Dehydratase