N-15 NMR RELAXATION STUDIES OF FREE AND INHIBITOR-BOUND 4-OXALOCROTONATE TAUTOMERASE - BACKBONE DYNAMICS AND ENTROPY CHANGES OF AN ENZYME UPON INHIBITOR BINDING

The solution secondary structure of 4-oxalocrotonate tautomerase (4-OT ), a 41 kDa homohexamer with 62 residues per subunit, consists of an a lpha-helix, two beta-strands, a beta-hairpin, two loops, two turns, an d a C-terminal coil [Stivers et al. (1996) Protein Sci, 5, 729-741]. T he general base, proline-1. as well as the two loops and the beta-hair pin have been shown to comprise the active site [Stivers et al. (1996) Biochemistry 35, 814-823]. The backbone dynamics of both the free enz yme and its complex with a substrate analog have been studied by H-1-d etected N-15 relaxation rates and NOE determinations at 500 and 600 MH z. Analysis of the data using the model-free formalism showed that the nanosecond to picosecond motion of 53 of the 60 backbone N-15-H vecto rs was highly restricted with a mean order parameter [S-2] = 0.87 +/- 0.03. The lowest backbone mobility (S-2 > 0.90) is found in the beta 1 -strand, loop 2, and turn 2. Greater backbone mobility is found in the active site (0.5 less than or equal to S-2 less than or equal to 0.83 ) and at C-terminal residues 58-62 (0.03 less than or equal to S-2 les s than or equal to 0.70), A tau(m) value for the flee hexamer of 13.7 ns at 42 degrees C was determined, consistent with a compact globular molecule of 41 kDa, Saturation of 4-OT with the analog of the dienolic intermediate and linear competitive inhibitor cis,cis-muconate (4) (K -D = 0.59 mM) increased the backbone S-2 of seven residues and decreas ed the backbone S-2 of another eight residues, both at the active site and at the antiparallel beta 1-beta 1 interface. The S-2 values of th e other 44 detectable NH vectors were not altered by the binding of 4. The increases in S-2, resulting from the ''freezing'' of the backbone NH vectors of seven residues upon the binding of 4, correspond to an unfavorable entropic contribution to Delta G(binding) of 3.2 +/- 1.1 k cal/mol. This freezing is partially compensated for by the mobilizatio n of the other eight residues, since the decreases in S-2 for these re sidues correspond to an entropic contribution to binding of -1.9 +/- 0 .1 kcal/mol. These entropy changes, resulting solely from alterations in high-frequency motion, are significant compared to the overall Delt a G(binding) = -4.6 kcal/mol for 4. Other effects of the binding of 4 include (1) changes in N-15 and NH chemical shifts localized to the ac tive site and (2) increases in the exchange contributions (R(ex)) to 1 /T-2 of backbone N-15 resonances at the active site and at the subunit inter face, reflecting microsecond to millisecond motions which may p lay a role in substrate binding (k(on) greater than or equal to 4 x 10 (6) M(-1) s(-1)) and/or catalysis (k(cat) = 10(3) s(-1)).