4-Oxalocrotonate tautomerase, a 41-kDa homohexamer: Backbone and side-chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy

4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated α-keto acids using Pro-1 as a general base (Stivers et al., 1996a, 1996b). We report the backbone and side-chain ¹H, ¹⁵N, and ¹³C NMR assignments and the solution secondary structure for 4-OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an α-helix (residues 13-30), two β-strands (β₁, residues 2-8; β₂, residues 39-45), a β-hairpin (residues 50-57), two loops (I, residues 9-12; II, 34-38), and two turns (I, residues 30-33; II, 47-50). The remaining residues form coils. The β₁ strand is parallel to the β₂ strand of the same subunit on the basis of cross strand NH_i-NH_j NOEs in a 2D ¹⁵N-edited ¹H-NOESY spectrum of hexameric 4-OT containing two ¹⁵N-labeled subunits/hexamer. The β₁ strand is also antiparallel to another β₁ strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from ¹⁵N T₁/T₂ measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe-50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2-oxo-1,6-hexanedioate. Modification of the general base, Pro-1, with the active site-directed irreversible inhibitor, 3-bromopyruvate, significantly alters the amide ¹⁵N and NH chemical shifts of residues in the β-hairpin and in loop II, providing evidence that these regions change conformation when the active site is occupied.