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 1 H, 15 N, and 13 C 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 ( β 1 , residues 2–8; β 2 , residues 39–45), a β ‐hairpin (residues 50–57), two loops (1, residues 9–12; II, 34–38), and two turns (I, residues 30–33; II, 47–50). The remaining residues form coils. The β 1 strand is parallel to the β 2 strand of the same subunit on the basis of cross strand NH i ‐NH j NOEs in a 2D 15 N‐edited 1 H‐NOESY spectrum of hexameric 4‐OT containing two 15 N‐labeled subunits/hexamer. The β 1 strand is also antiparallel to another β 1 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 15 N T 1 / T 2 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‐l, 6‐hexanedioate. Modification of the general base, Pro‐1, with the active site‐directed irreversible inhibitor, 3‐bromopyruvate, significantly alters the amide 15 N 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.