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 re sidues per subunit, catalyzes the isomerization of unsaturated oc-keto acids using Pro-1 as a general base (Stivers et al., 1996a, 1996b). W e report the backbone and side-chain H-1, N-15, and C-13 NMR assignmen ts and the solution secondary structure for 4-OT using 2D and 3D homon uclear and heteronuclear NMR methods. The subunit secondary structure consists of an alpha-helix (residues 13-30), two beta-strands (beta(1) , residues 2-8; beta(2), residues 39-45), a beta-hairpin (residues 50- 57), two loops (I, residues 9-12; II, 34-38), and two turns (I, residu es 30-33; II, 47-50). The remaining residues form coils. The beta(1) s trand is parallel to the beta(2) strand of the same subunit on the bas is of cross strand NHi-NHj NOEs in a 2D N-15-edited H-1-NOESY spectrum of hexameric 4-OT containing two N-15-labeled subunits/hexamer. The b eta(1) strand is also antiparallel to another beta(1) strand from an a djacent subunit forming a subunit interface. Because only three such p airwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and th e rotational correlation time (14.5 ns) estimated from N-15 T-1/T-2 me asurements, are consistent with the hexameric molecular weight of 41 k Da. Residue Phe-50 is in the active site on the basis of transferred N OEs to the bound partial substrate 2-oxo-1,6-hexanedioate. Modificatio n of the general base, Pro-1, with the active site-directed irreversib le inhibitor, 3-bromopyruvate, significantly alters the amide N-15 and NH chemical shifts of residues in the beta-hairpin and in loop II, pr oviding evidence that these regions change conformation when the activ e site is occupied.