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15N NMR relaxation studies of free and inhibitor-bound 4-oxalocrotonate tautomerase: backbone dynamics and entropy changes of an enzyme upon inhibitor binding.
Biochemistry. 1996 Dec 17; 35(50):16036-47.B

Abstract

The solution secondary structure of 4-oxalocrotonate tautomerase (4-OT), a 41 kDa homohexamer with 62 residues per subunit, consists of an alpha-helix, two beta-strands, a beta-hairpin, two loops, two turns, and a C-terminal coil [Stivers et al. (1996) Protein Sci. 5, 729-741]. The general base, proline-1, as well as the two loops and the beta-hairpin have been shown to comprise the active site [Stivers et al. (1996) Biochemistry 35, 814-823]. The backbone dynamics of both the free enzyme and its complex with a substrate analog have been studied by 1H-detected 15N relaxation rates and NOE determinations at 500 and 600 MHz. Analysis of the data using the model-free formalism showed that the nanosecond to picosecond motion of 53 of the 60 backbone 15N-H vectors was highly restricted with a mean order parameter mean value of S2 = 0.87 +/- 0.03. The lowest backbone mobility (S2 > 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 < or = S2 < or = 0.83) and at C-terminal residues 58-62 (0.03 < or = S2 < or = 0.70). A tau m value for the free 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) (KD = 0.59 mM) increased the backbone S2 of seven residues and decreased the backbone S2 of another eight residues, both at the active site and at the antiparallel beta 1-beta 1 interface. The S2 values of the other 44 detectable NH vectors were not altered by the binding of 4. The increases in S2, 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 Gbinding of 3.2 +/- 1.1 kcal/mol. This freezing is partially compensated for by the mobilization of the other eight residues, since the decreases in S2 for these residues 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 delta Gbinding = -4.6 kcal/mol for 4. Other effects of the binding of 4 include (1) changes in 15N and NH chemical shifts localized to the active site and (2) increases in the exchange contributions (R(ex)) to 1/T2 of backbone 15N resonances at the active site and at the subunit interface, reflecting microsecond to millisecond motions which may play a role in substrate binding (k(on) > or = 4 x 10(6) M-1 s-1) and/or catalysis (kcat = 10(3) s-1).

Authors+Show Affiliations

Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205-2185, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8973173

Citation

Stivers, J T., et al. "15N NMR Relaxation Studies of Free and Inhibitor-bound 4-oxalocrotonate Tautomerase: Backbone Dynamics and Entropy Changes of an Enzyme Upon Inhibitor Binding." Biochemistry, vol. 35, no. 50, 1996, pp. 16036-47.
Stivers JT, Abeygunawardana C, Mildvan AS. 15N NMR relaxation studies of free and inhibitor-bound 4-oxalocrotonate tautomerase: backbone dynamics and entropy changes of an enzyme upon inhibitor binding. Biochemistry. 1996;35(50):16036-47.
Stivers, J. T., Abeygunawardana, C., & Mildvan, A. S. (1996). 15N NMR relaxation studies of free and inhibitor-bound 4-oxalocrotonate tautomerase: backbone dynamics and entropy changes of an enzyme upon inhibitor binding. Biochemistry, 35(50), 16036-47.
Stivers JT, Abeygunawardana C, Mildvan AS. 15N NMR Relaxation Studies of Free and Inhibitor-bound 4-oxalocrotonate Tautomerase: Backbone Dynamics and Entropy Changes of an Enzyme Upon Inhibitor Binding. Biochemistry. 1996 Dec 17;35(50):16036-47. PubMed PMID: 8973173.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 15N NMR relaxation studies of free and inhibitor-bound 4-oxalocrotonate tautomerase: backbone dynamics and entropy changes of an enzyme upon inhibitor binding. AU - Stivers,J T, AU - Abeygunawardana,C, AU - Mildvan,A S, PY - 1996/12/17/pubmed PY - 1996/12/17/medline PY - 1996/12/17/entrez SP - 16036 EP - 47 JF - Biochemistry JO - Biochemistry VL - 35 IS - 50 N2 - The solution secondary structure of 4-oxalocrotonate tautomerase (4-OT), a 41 kDa homohexamer with 62 residues per subunit, consists of an alpha-helix, two beta-strands, a beta-hairpin, two loops, two turns, and a C-terminal coil [Stivers et al. (1996) Protein Sci. 5, 729-741]. The general base, proline-1, as well as the two loops and the beta-hairpin have been shown to comprise the active site [Stivers et al. (1996) Biochemistry 35, 814-823]. The backbone dynamics of both the free enzyme and its complex with a substrate analog have been studied by 1H-detected 15N relaxation rates and NOE determinations at 500 and 600 MHz. Analysis of the data using the model-free formalism showed that the nanosecond to picosecond motion of 53 of the 60 backbone 15N-H vectors was highly restricted with a mean order parameter mean value of S2 = 0.87 +/- 0.03. The lowest backbone mobility (S2 > 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 < or = S2 < or = 0.83) and at C-terminal residues 58-62 (0.03 < or = S2 < or = 0.70). A tau m value for the free 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) (KD = 0.59 mM) increased the backbone S2 of seven residues and decreased the backbone S2 of another eight residues, both at the active site and at the antiparallel beta 1-beta 1 interface. The S2 values of the other 44 detectable NH vectors were not altered by the binding of 4. The increases in S2, 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 Gbinding of 3.2 +/- 1.1 kcal/mol. This freezing is partially compensated for by the mobilization of the other eight residues, since the decreases in S2 for these residues 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 delta Gbinding = -4.6 kcal/mol for 4. Other effects of the binding of 4 include (1) changes in 15N and NH chemical shifts localized to the active site and (2) increases in the exchange contributions (R(ex)) to 1/T2 of backbone 15N resonances at the active site and at the subunit interface, reflecting microsecond to millisecond motions which may play a role in substrate binding (k(on) > or = 4 x 10(6) M-1 s-1) and/or catalysis (kcat = 10(3) s-1). SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8973173/15N_NMR_relaxation_studies_of_free_and_inhibitor_bound_4_oxalocrotonate_tautomerase:_backbone_dynamics_and_entropy_changes_of_an_enzyme_upon_inhibitor_binding_ L2 - https://doi.org/10.1021/bi961834q DB - PRIME DP - Unbound Medicine ER -