Catalytic role of the amino-terminal proline in 4-oxalocrotonate tautomerase: Affinity labeling and heteronuclear NMR studies

4-Oxalocrotonate tautomerase (EC 5.3.2-; 4-OT), a hexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated α-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., and Wigley, D. B. (1996) Biochemistry 35, 792802]. 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-I (δ = 49.2 ppm at pH 6.40 and 42°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- ¹³C-labeled lactyl adduct derived from [3-¹³C]bromopyruvate was then directly demonstrated using two heteronuclear NMR methods, an ¹H-¹³C HSQC experiment and a novel inverse correlation experiment which we call H(C)N. The chemical shift of the modified ¹⁵N resonance (δ = 86.5 ppm) is consistent with that of an alkylated and cationic, aminoterminal proline. Affinity labeling with 2-¹⁴C-labeled bromopyruvate indicates that the ultimate stoichiometry of modification is 1 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 k(inact)/K₁ for affinity labeling by 3-BP yields a pK(a) value of 6.7 ± 0.3, in reasonable agreement with the pK(a) values found for k(cat)/K(M) for the non-sticky substrate 2-hydroxy-2,4-pentadienoate and by direct NMR titration of Pro-I [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814-823]. These results strongly implicate the amino-terminal proline as the general- base catalyst on 4-OT.