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PDBsum entry 4lht
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Oxidoreductase
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PDB id
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4lht
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References listed in PDB file
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Key reference
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Title
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P450cin active site water: implications for substrate binding and solvent accessibility.
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Authors
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Y.Madrona,
S.A.Hollingsworth,
B.Khan,
T.L.Poulos.
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Ref.
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Biochemistry, 2013,
52,
5039-5050.
[DOI no: ]
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PubMed id
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Abstract
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In P450cin, Tyr81, Asp241, Asn242, two water molecules, and the substrate
participate in a complex H-bonded network. The role of this H-bonded network in
substrate binding and catalysis has been probed by crystallography,
spectroscopy, kinetics, isothermal titration calorimetry (ITC), and molecular
dynamics. For the Y81F mutant, the substrate binds about 20-fold more weakly and
Vmax decreases by about 30% in comparison to WT. The enhanced susceptibility of
the heme to H2O2-mediated destruction in Y81F suggests that this mutant favors
the open, low-spin conformational state. Asn242 H-bonds directly with the
substrate, and replacing this residue with Ala results in water taking the place
of the missing Asn side chain. This mutant exhibits a 70% decrease in activity.
Crystal structures and molecular dynamics simulations of substrate-bound
complexes show that the solvent has more ready access to the active site,
especially for the N242A mutant. This accounts for about a 64% uncoupling of
electron transfer from substrate hydroxylation. These data indicate the
importance of the interconnected water network on substrate binding and on the
open/closed conformational equilibrium, which are both critically important for
maintaining high-coupling efficiency.
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