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Title:Molecular specificity of substrate recognition and activation in cytochrome P-450(CAM)
Author(s):Loida, Paul John
Doctoral Committee Chair(s):Sligar, Stephen G.
Department / Program:Chemistry, Biochemistry
Biophysics, General
Discipline:Chemistry, Biochemistry
Biophysics, General
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Biophysics, General
Abstract:The mechanisms by which biological macromolecules recognize small molecules are of fundamental relevance to the maintenance of living systems and the chemistry of noncovalent bonding. Hydrogen bonding and hydrophobic interactions have been implicated in binding and activation of hydrocarbon substrates by cytochrome P-450$\sb{\rm cam}$. The dependence of the reaction efficiency and specificity on the structural complementarity in the P-450$\sb{\rm cam}$-substrate complex suggests that the active site structure is of paramount importance in mediating catalysis. Cytochrome P-450$\sb{\rm cam}$ was used as a model system to elucidate the role of specific active site residues in substrate recognition and to engineer enzyme-substrate interactions for novel specificities.
Active site mutations of hydrophobic and hydrogen bonding residues were designed to alter the efficiency and specificity of aliphatic hydroxylation by changing the position and orientation of the substrate in the active site. Steric effects were assessed by engineering hydrophobic side chains at multiple sites around the perimeter of the binding pocket such that the access of the substrate to the heme is predicted to be either inhibited or enhanced. Ethylbenzene and a series of chlorinated ethanes were used to show that the position of steric bulk in the active site is correlated with the catalytic efficiency of the enzyme under both oxidative and reductive reaction conditions. Thus, the orientation of the substrate in the active site was altered in a predictable manner by the modulating the topology of the active site cavity. The uncoupling of reducing equivalents was strongly dependent on the oxidase branch point in the reaction, indicating that reaction efficiency at the level of the putative (FeO) $\sp{3+}$ is controlled by substrate access to the iron. Polar contributions to substrate recognition were evaluated by the design of a hydrogen bond switch, which effected the expected changes in hydroxylation regiospecificity with (1R)- and(1S)-norcamphor. In general, active site design of hydrophobic contacts and polar interactions resulted in the expected perturbations in the P-450$\sb{\rm cam}$ catalyzed reaction, suggesting that protein engineering for enhanced enzyme activity and specificity is a viable approach to the pursuit of novel catalysts.
Issue Date:1994
Rights Information:Copyright 1994 Loida, Paul John
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9512467
OCLC Identifier:(UMI)AAI9512467

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