Conformational and allosteric changes in fructose 1,6-bisphosphatase upon photoaffinity labeling with 2-azidoadenosine monophosphate.
The photoaffinity analog 2-azido-AMP was found to be a potent allosteric inhibitor of pig kidney fructose 1,6-bisphosphatase. UV-induced covalent incorporation of 2-azido-[8-3H]AMP fully inactivated the enzyme at a level stoichiometric with its subunit composition (4 mol of analog/mol of tetramer). The photoincorporation and inactivation were prevented by the presence of AMP but not by the substrate, IMP, or adenosine. Enzyme fully modified with 2-azido-AMP was capable of binding fructose 1,6-bisphosphate but not AMP. The analog thus specifically modified the enzyme's allosteric sites. Titrations of the native enzyme's fast reacting cysteines with 5,5'-dithiobis-(2-nitrobenzoic acid) distinguished four conformational states of the enzyme: E(+/- Mg2+), E(AMP), E(AMP)Mg2+, E(Fru-1,6-P2)x. AMP had a biphasic effect with a low affinity phase that could be mimicked by IMP, a competitive inhibitor of fructose 1,6-bisphosphate binding. Enzyme fully modified with 2-azido-AMP was isolated and observed to have a similar conformation and to undergo similar ligand-induced conformational changes as the native enzyme-AMP complex. Random modification of 50% of the subunits with 2-azido-AMP shifted the AMP inhibition curve from sigmoidal to hyperbolic without changing the inhibition constant. AMP-induced cooperativity in the pig kidney fructose 1,6-bisphosphatase appears to be restricted to 2 subunits without alterations in AMP affinity.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000494:Allosteric Regulation; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013552:Swine
D000345:Affinity Labels; D001386:Azides; D000249:Adenosine Monophosphate; C018838:2-azidoadenosine 5'-monophosphate; D006597:Fructose-Bisphosphatase