4-Oxalocrotonate Tautomerase:  pH Dependence of Catalysis and p<i>K</i><sub>a</sub> Values of Active Site Residues

Stivers, James T.; Abeygunawardana, Chitrananda; Mildvan, Albert S.; Hajipour, Gholamhossein; Whitman, Christian P.

doi:10.1021/bi9510789

Cited by 123 publications

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“…A pK, value of 6.4 for Pro-I was determined by "N-NMR titration and this pK, is 3 units lower than the model compound proline amide (Stivers et al, 1996a), most likely the result of an active site of low dielectric constant. The pH dependenceof the reaction rate (Stivers et al, 1996a) also suggested a requirement for an enzymic general acid (AH, Fig. 1) with a pK, of 9.0, to stabilize the developing negative charge on the carbonyl oxygen (Xue et al, 1990;Gerlt & Gassman, 1993).…”

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confidence: 94%

“…1) is the catalytic base (Stivers et al, 1996a(Stivers et al, , 1996b. A pK, value of 6.4 for Pro-I was determined by "N-NMR titration and this pK, is 3 units lower than the model compound proline amide (Stivers et al, 1996a), most likely the result of an active site of low dielectric constant. The pH dependenceof the reaction rate (Stivers et al, 1996a) also suggested a requirement for an enzymic general acid (AH, Fig.…”

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confidence: 99%

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confidence: 99%

“…CF600 (Subtion; TSP, sodium 3-(trimethylsilyl)propionate-2,2,3,3,-d4. ramanya et al, 1996) shows that the enzyme consists of a 729 dimensional; 3D, three-dimensional; RF, radiofrequency; TOCSY, total 409866* * 1'9-A reso1ution crysta1 structure Of a 73v0 homo1- Catalytic mechanism of 4-oxalocrotonate tautomerase (Stivers et al, 1996a(Stivers et al, . 1996b.…”

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confidence: 99%

“…Recent NMR and mechanistic studies of 4-OT have established that the amino-terminal proline residue ( Fig. 1) is the catalytic base (Stivers et al, 1996a(Stivers et al, , 1996b. A pK, value of 6.4 for Pro-I was determined by "N-NMR titration and this pK, is 3 units lower than the model compound proline amide (Stivers et al, 1996a), most likely the result of an active site of low dielectric constant.…”

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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

Stivers¹,

Abeygunawardana²,

Whitman³

et al. 1996

Protein Science

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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.

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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

Stivers¹,

Abeygunawardana²,

Whitman³

et al. 1996

Protein Science

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

et al. 1999

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Protein Flexibility in In Silico Screening

Gohlke

Kopitz

2010

Burger's Medicinal Chemistry and Drug Discovery

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We summarize computational approaches in structure‐based ligand design (SBLD) and in silico screening that address issues of protein flexibility and mobility. In particular, we consider how protein plasticity can be incorporated into docking strategies. As a first requirement, one needs to detect what can move and how. Moving protein parts can be identified from experimental information as well as established computational techniques such as molecular dynamics (MD) simulations, graph theoretical and geometry‐based approaches, or harmonic analysis‐based methods. Second, this knowledge needs to be transformed into a docking algorithm. A multitude of approaches considering protein mobility has been introduced recently, with motions modeled either implicitly or explicitly. In the latter case, one can further distinguish between modeling of side‐chain‐only motions and motions including backbone changes. In all cases, accuracy needs to be balanced against efficiency. Case studies for which the inclusion of protein plasticity was crucial to success are noted along these lines. This allows us to identify scope and limitations of the current approaches, as well as guidelines for further developments.

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4-Oxalocrotonate Tautomerase: pH Dependence of Catalysis and pK_a Values of Active Site Residues

Cited by 123 publications

References 35 publications

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, a 41‐kDa homohexamer: Backbone and side‐chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy

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

Protein Flexibility in In Silico Screening

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4-Oxalocrotonate Tautomerase: pH Dependence of Catalysis and pKa Values of Active Site Residues

Cited by 123 publications

References 35 publications

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, a 41‐kDa homohexamer: Backbone and side‐chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy

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

Protein Flexibility in In Silico Screening

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Product

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4-Oxalocrotonate Tautomerase: pH Dependence of Catalysis and pK_a Values of Active Site Residues