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

Abstract

Abstract4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated a-keto acids using Pro-1 as a general base (Stivers et al., 1996a(Stivers et al., , 1996b. We report the backbone and side-chain 'H, "N, and I3C 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 a-helix (residues 13-30), two 0-strands (PI, residues 2-8; &, residues 39-45), a @-hairpin (residues 50-57), two loops (I, residues 9-12; 11, 34-38), and two turns (I, residues 30-33; II,47-50). The remaining residues form coils. The PI strand is parallel to the pz strand of the same subunit on the basis of cross strand NHi-NHj NOEs in a 2D "N-edited 'H-NOESY spectrum of hexameric 4-OT containing two I5N-labeled subunits/hexamer. The 0' strand is also antiparallel to another PI 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 I5N T l / T , 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 2-0~0-1,6-hexanedioate. Modification of the general base, Pro-1, with the active site-directed irreversible inhibitor, 3-bromopyruvate, significantly alters the amide I5N and NH chemical shifts of residues in the 0-hairpin and in loop 11, providing evidence that these regions change conformation when the active site is occupied.

Characterizing the Use of Perdeuteration in NMR Studies of Large Proteins:13C,15N and1H Assignments of Human Carbonic Anhydrase II

“…The study of increasingly larger proteins has been made possible by advances in NMR spectrometer design, multi-dimensional and multi-nuclear NMR pulse sequences Muhandiram & Kay, 1994), and 13 C/ 15 N isotope labeling techniques Venters et al, 1991). Resonance assignments have been reported for two serine proteases of molecular mass 27 kDa and 28 kDa (Fogh et al, 1994;Remerowski et al, 1994) and for 4-oxalocrotonate tautomerase, a 41 kDa homohexamer (Stivers et al, 1996). In the latter case, NMR spectra were taken at a relatively high temperature (42°C) and at a relatively high subunit concentration (3.5 to 7.9 mM).…”

Docking of 4-oxalocrotonate tautomerase substrates: Implications for the catalytic mechanism

“…They span a relatively wide range from 10.3 Ϯ 0.2 to 9.0 Ϯ 0.3 for the 4OT wild type 4 to 9.5 Ϯ 0.2 and 9.7 Ϯ 0.2 for 4OT mutants where Pro1 were replaced by Gly or Ala, respectively. 31 Our electrostatic calculations indicate that among the ionizable active site residues, only Arg39 and Lys47 have calculated pK a values suitable to match these experimental values (Table III). The calculated values for these two residues agree better with the experimental value obtained for 4OT using 2-hydroxymuconate, 10.3 Ϯ 0.2.…”

“…This result is supported by nmr measurement indicating large changes in the amide proton chemical shifts of Arg39 and other residues in the loop preceding Arg39 upon covalent binding of 3-bro- mopyruvate to Pro1. 7,31 This suggests a conformational change in the 4OT active site resulting from the direct interaction between the C1-carboxylate group of this substrate-like ligand and the guanidinium of Arg39. So far, there is no evidence of any conformational changes that would place the Lys47 side chain into the active site.…”

“…1OTM will be taken as the reference structure for this discussion since it corresponds to the wild type enzyme for which all the experimental pK a measurements were performed. 4,31 The theoretical and experimental pK a values are for the free enzymes.…”

Secondary structure of ?-hydroxydecanoyl thiol ester dehydrase, a 39-kDa protein, derived from H?, C?, C? and CO signal assignments and the Chemical Shift Index: Comparison with the crystal structure

“…After submitting this manuscript, the assignments and secondary structure of 4-oxalocrotonate tautomerase, a 41-kDa homohexamer containing 6 • 62 = 372 amino acid residues, were reported (Stivers et al, 1996).…”