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http://purl.uniprot.org/citations/8547260http://www.w3.org/1999/02/22-rdf-syntax-ns#typehttp://purl.uniprot.org/core/Journal_Citation
http://purl.uniprot.org/citations/8547260http://www.w3.org/1999/02/22-rdf-syntax-ns#typehttp://purl.uniprot.org/core/Journal_Citation
http://purl.uniprot.org/citations/8547260http://www.w3.org/1999/02/22-rdf-syntax-ns#typehttp://purl.uniprot.org/core/Citation
http://purl.uniprot.org/citations/8547260http://www.w3.org/2000/01/rdf-schema#comment"4-Oxalocrotonate tautomerase (EC 5.3.2-; 4-OT), a hexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids, converting unconjugated ketones to the conjugated isomers via a dienolic intermediate. The recently solved crystal structure of an isozyme of 4-OT suggests that the amino-terminal proline is the catalytic base [Subramanya, H. S., Roper, D. I., Dauter, Z., Dodson, E. J., Davies, G. J., Wilson, K. S., & Wigley, D. B. (1996) Biochemistry 35, 792-802]. In support of this proposed role, we have found that the active-site-directed irreversible inhibitor 3-bromopyruvate (3-BP) blocks the amino terminus of 4-OT to Edman degradation and results in the disappearance of the 15N resonance of Pro-1 (delta = 49.2 ppm at pH 6.40 and 42 degrees C) in the 15N NMR spectrum of uniformly 15N-labeled 4-OT. Furthermore, covalent bonding between a 15N resonance of 4-OT and the methylene carbon of the reduced, 3-(13)C-labeled lactyl adduct derived from [3-(13)C]-bromopyruvate was then directly demonstrated using two heteronuclear NMR methods, an 1H-(13)C HSQC experiment and a novel inverse correlation experiment which we call H(C)N. The chemical shift of the modified 15N resonance (delta = 86.5 ppm) is consistent with that of an alkylated and cationic, amino-terminal proline. Affinity labeling with 2-(14)C-labeled bromopyruvate indicates that the ultimate stoichiometry of modification is I equiv of 3-BP per 4-OT monomer. However, an analysis of the residual enzyme activity after differing extents of fractional modification with 3-BP indicates that modification of three active sites per hexamer abolishes essentially all activity of the hexamer. Thus, 4-OT exhibits half-of-the-sites stoichiometry with 3-BP. Finally, the pH dependence of kinact/KI for affinity labeling by 3-BP yields a pKa value of 6.7 +/-0.3, in reasonable agreement with the pKa values found for kcat/KM for the non-sticky substrate 2-hydroxy-2,4-pentadienoate and by direct NMR titration of Pro-1 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., & Whitman, C. P. (1996) Biochemistry 35, 814-823]. These results strongly implicate the amino-terminal proline as the general-base catalyst on 4-OT."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.org/dc/terms/identifier"doi:10.1021/bi951077g"xsd:string
http://purl.uniprot.org/citations/8547260http://purl.org/dc/terms/identifier"doi:10.1021/bi951077g"xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Abeygunawardana C."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Abeygunawardana C."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Chen L.H."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Chen L.H."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Hajipour G."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Hajipour G."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Mildvan A.S."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Mildvan A.S."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Stivers J.T."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Stivers J.T."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Whitman C.P."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/author"Whitman C.P."xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/date"1996"xsd:gYear
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/date"1996"xsd:gYear
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/name"Biochemistry"xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/name"Biochemistry"xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/pages"803-813"xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/pages"803-813"xsd:string
http://purl.uniprot.org/citations/8547260http://purl.uniprot.org/core/title"Catalytic role of the amino-terminal proline in 4-oxalocrotonate tautomerase: affinity labeling and heteronuclear NMR studies."xsd:string
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