Angle selective ENDOR of nitroxyl spin-labels is briefly reviewed to illustrate the methodology of structure analysis developed in our laboratory for characterizing catalytically competent intermediates of enzyme catalyzed reactions. ENDOR structure determination of a reaction intermediate of alpha-chymotrypsin formed with a kinetically specific spin-labeled substrate and of an enzyme-inhibitor complex formed with a spin-labeled transition-state inhibitor analog is briefly described. Both spin-labeled molecules bound in the active site of the enzyme are found in torsionally distorted conformations. It is suggested that this torsionally distorted state in which the bound ligand is of higher potential energy than in the ground state conformation reflects substrate destabilization in the course of the enzyme catalyzed reaction.
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