Active site mechanics of liver microsomal cytochrome P-450

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Abstract

For a set of 10 para-substituted toluene derivatives, three enzymatic constants were determined describing their interaction with purified rabbit liver microsomal P-450LM2. The three constants were the catalytic rate constant (kcat) for hydroxylation, the apparent dissociation constant (Kd) for the enzyme-substrate complex, and the interaction energy (ΔGint) between the substrate-binding and spin-state equilibria. The para-substituents of the toluene substrates were: hydrogen, fluoro, bromo, chloro, iodo, nitro, methyl, cyano, isopropyl, and t-butyl. Linear free energy correlations were sought between the enzymatic constants and several physical constants of the individual substrate molecules. These correlations would be useful both for empirical prediction purposes and for insight into active site chemistry and mechanics. Catalytic rates were correlated by a linear combination of the Hansch π hydrophobic constant and the Hammett σ value. A deuterium isotope effect (DV) of 2.6 for d8-toluene compared to d0-toluene confirmed that hydrogen abstraction was partially rate-limiting with this series of substrates. Apparent dissociation constants were predicted by a linear combination of the molar volume and π, while the spin-state interaction energies were best predicted by a linear combination of the Hansch π hydrophobic constant and the reciprocal of the dielectric constant.

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    This research was supported by Research Grants GM28737 and ESO3600 from the United States Public Health Service and by the University of Connecticut Research Foundation. A preliminary report of part of this article has been presented (1).

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