STRUCTURAL REQUIREMENTS FOR THE BINDING OF AMP TO THE ALLOSTERIC SITE OF NAD-SPECIFIC ISOCITRATE DEHYDROGENASE FROM BAKERS-YEAST

The specificity of yeast NAD-specific isocitrate dehydrogenase for the structures of the allosteric effector 5′-AMP was examined with analogues modified in the purine ring, pentosyl group, and 5′-phosphate group. An unsubstituted 6-amino group was essential for activation as was the phosphoryl group at the 5′-position. Activity was retained when an oxygen function of the 5′-phosphoryl was replaced by sulfur (Murry & Atkinson, 1968) or by nitrogen (phosphoramidates). 2-NH2-AMP, 2-azido-AMP, and 8-NH2-AMP were active; 8-azido-AMP and 8-Br-AMP were inactive. The configuration or nature of sub-stituents about carbons 2′ and 3′ of the pentosyl portion of AMP was not critical for allosteric activation since AMP analogues containing, e.g., 2′,3′-dideoxyribose or the bulky 2′,3′-O-(2,4,6-trinitrocyclo-hexadienylidene) substituent (TNP-AMP) were active. TNP-AMP was bound to the enzyme with fluorescence enhancement and had an S0.5 for activation similar to the S0.5 for AMP. Positive effector activity was decreased when the pentosyl moiety of 5′-AMP was replaced by the six-membered nitrogen-containing morpholine group, indicating that the pentosyl group may be critical as a spacer for the proper geometry of binding to enzyme at the 6-amino and 5′-phosphoryl groups of 5′-AMP. A comparison of molecular models of 5′-AMP with 8,5′-cycloAMP suggests that the species of 5′-AMP required for binding to the enzyme contains the purine and ribose moieties in an anti conformation and positioning of the 5′-phosphate trans with respect to carbon 4′. This is consistent with the finding that (S)-8,5′-cycloAMP was a potent negative allosteric modifier (i.e., it increased Km for isocitrate) whose effect could be reversed competitively by 5′-AMP, whereas the R epimer was inactive. © 1990, American Chemical Society. All rights reserved.