Half-of-the-Sites Binding of Reactive Intermediates and Their Analogues to 4-Oxalocrotonate Tautomerase and Induced Structural Asymmetry of the Enzyme

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• 作者：Hugo F. Azurmendi ; Scott G. Miller ; Christian P. Whitman ; and Albert S. Mildvan
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：May 31, 2005
• 年：2005
• 卷：44
• 期：21
• 页码：7725 - 7737
• 全文大小：1085K
• 年卷期：v.44,no.21(May 31, 2005)
• ISSN：1520-4995

文摘

4-Oxalocrotonate tautomerase (4-OT), a homohexameric enzyme, converts the unconjugatedenone, 2-oxo-4-hexenedioate (1), to the conjugated enone, 2-oxo-3-hexenedioate (3), via a dienolicintermediate, 2-hydroxymuconate (2). Pro-1 serves as the general base, and both Arg-11 and Arg-39function in substrate binding and catalysis in an otherwise hydrophobic active site. Although 4-OT exhibitshyperbolic kinetics and no structural asymmetry either by X-ray or by NMR, inactivation by two affinitylabels 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 (s/entities/ge.gif">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 backboneNH and of Arg Ns/gifchars/epsilon.gif" BORDER=0 >H resonances and one-dimensional ¹⁵N NMR titrations of Arg Ns/gifchars/epsilon.gif" BORDER=0 > resonances. Alltitrations showed substoichiometric binding of the equilibrium mixtures to 3 ± 1 sites per hexamer withapparent dissociation constants comparable to the K_m values of the intermediates. Compound 4 also bound1 order of magnitude less tightly at another site, suggesting negative cooperativity. Consistent with negativecooperativity, asymmetry of the resulting complexes at saturating levels of 2 and 4 is indicated by splittingof the backbone NH resonances of 11 residues and 10 residues of 4-OT, respectively. The dicarboxylatecompetitive inhibitor, (2E)-fluoromuconate (5), with a K_I of 45 ± 7 s/entities/mgr.gif">M, also exhibited substoichiometricbinding to 3 ± 1 sites per hexamer, with a K_D of 25 ± 18 s/entities/mgr.gif">M, and splitting of the backbone NH resonanceof L8. The monocarboxylate inhibitors (2E)- (6) and (2Z)-2-fluoro-2,4-pentadienoate (7) showed muchweaker binding (K_D = 3.1 ± 1.3 mM), as well as splitting of two and five backbone NH resonances,respectively, indicating asymmetry of the complexes. The Ns/gifchars/epsilon.gif" BORDER=0 > resonances of both Arg-11 and Arg-39were shifted downfield, and that of Pro-1N was broadened by all ligands, consistent with the major catalyticroles of these residues. Structural pathways for the site-site interactions which result in negativecooperativity are proposed on the basis of the X-ray structures of free and affinity-labeled 4-OT. Selectiveresonance broadenings induced by the binding of inactive analogues and active intermediates indicateresidues which may be mobilized during reversible ligand binding and during catalysis, respectively.