Half-of-the-sites binding of reactive intermediates and their analogues to 4-oxalocrotonate tautomerase and induced structural asymmetry of the enzyme.

4-Oxalocrotonate tautomerase (4-OT), a homohexameric enzyme, converts the unconjugated enone, 2-oxo-4-hexenedioate (1), to the conjugated enone, 2-oxo-3-hexenedioate (3), via a dienolic intermediate, 2-hydroxymuconate (2). Pro-1 serves as the general base, and both Arg-11 and Arg-39 function in substrate binding and catalysis in an otherwise hydrophobic active site. Although 4-OT exhibits hyperbolic kinetics and no structural asymmetry either by X-ray or by NMR, inactivation by two affinity labels showed half-site stoichiometry [Stivers, J. T., et al. (1996) Biochemistry 35, 803-813; Johnson, W. H., Jr., et al. (1997) Biochemistry 36, 15724-15732], and titration of the R39Q mutant with cis,cis-muconate showed negative cooperativity [Harris, T. K., et al. (1999) Biochemistry 38, 12343-12357]. To test for anticooperativity during catalysis, 4-OT was titrated with equilibrium mixtures (> or = 81% product) of the reactive dicarboxylate or monocarboxylate intermediates, 2 or 2-hydroxy-2,4-pentadienoate (4), respectively, in three types of NMR experiments: two-dimensional 1H-15N HSQC titrations of backbone NH and of Arg N epsilonH resonances and one-dimensional 15N NMR titrations of Arg N epsilon resonances. All titrations showed substoichiometric binding of the equilibrium mixtures to 3 +/- 1 sites per hexamer with apparent dissociation constants comparable to the Km values of the intermediates. Compound 4 also bound 1 order of magnitude less tightly at another site, suggesting negative cooperativity. Consistent with negative cooperativity, asymmetry of the resulting complexes at saturating levels of 2 and 4 is indicated by splitting of the backbone NH resonances of 11 residues and 10 residues of 4-OT, respectively. The dicarboxylate competitive inhibitor, (2E)-fluoromuconate (5), with a KI of 45 +/- 7 microM, also exhibited substoichiometric binding to 3 +/- 1 sites per hexamer, with a KD of 25 +/- 18 microM, and splitting of the backbone NH resonance of L8. The monocarboxylate inhibitors (2E)- (6) and (2Z)-2-fluoro-2,4-pentadienoate (7) showed much weaker binding (KD = 3.1 +/- 1.3 mM), as well as splitting of two and five backbone NH resonances, respectively, indicating asymmetry of the complexes. The N epsilon resonances of both Arg-11 and Arg-39 were shifted downfield, and that of Pro-1N was broadened by all ligands, consistent with the major catalytic roles of these residues. Structural pathways for the site-site interactions which result in negative cooperativity are proposed on the basis of the X-ray structures of free and affinity-labeled 4-OT. Selective resonance broadenings induced by the binding of inactive analogues and active intermediates indicate residues which may be mobilized during reversible ligand binding and during catalysis, respectively.