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Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes
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P450cin active site water: implications for substrate binding and solvent accessibility.

Yarrow Madrona1, Scott A Hollingsworth, Bushra Khan

  • 1Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, United States.

Biochemistry
|July 9, 2013
PubMed
Summary
This summary is machine-generated.

The intricate hydrogen-bonded network involving water molecules and specific amino acids in P450cin is crucial for substrate binding and efficient catalysis. Disrupting this network significantly impairs enzyme activity and substrate affinity.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Cytochrome P450cin (P450cin) utilizes a hydrogen-bonded network involving tyrosine 81 (Tyr81), aspartate 241 (Asp241), asparagine 242 (Asn242), and water molecules for substrate interaction and catalysis.
  • Understanding the role of this intricate network is key to elucidating P450cin's catalytic mechanisms and efficiency.

Purpose of the Study:

  • To investigate the functional significance of the hydrogen-bonded network in P450cin's active site.
  • To determine the impact of mutations within this network on substrate binding, catalytic activity, and conformational dynamics.

Main Methods:

  • Site-directed mutagenesis (Y81F, N242A) to probe specific residue roles.
  • Enzyme kinetics (Vmax) and substrate binding affinity measurements.
  • Isothermal titration calorimetry (ITC) for binding thermodynamics.
  • X-ray crystallography and molecular dynamics (MD) simulations for structural insights.
  • Spectroscopy to assess heme susceptibility to oxidative damage.

Main Results:

  • The Y81F mutant exhibited a 20-fold decrease in substrate binding affinity and a 30% reduction in Vmax compared to wild-type (WT).
  • The Y81F mutant showed increased susceptibility to H₂O₂-mediated heme destruction, indicating a shift towards an open, low-spin state.
  • The N242A mutant displayed a 70% decrease in activity, with water molecules occupying the space of the Asn242 side chain.
  • MD simulations and crystal structures revealed increased solvent access to the active site in the N242A mutant, leading to a 64% uncoupling of electron transfer from hydroxylation.

Conclusions:

  • The interconnected water network and specific amino acid residues (Tyr81, Asn242) are critical for efficient substrate binding and catalysis in P450cin.
  • This network influences the active site's conformational equilibrium (open/closed states), which is vital for maintaining high catalytic coupling efficiency.
  • Mutations disrupting the hydrogen-bonded network lead to reduced substrate affinity, impaired catalysis, and altered conformational dynamics, highlighting the network's essential role.