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|Title:||Bioorganic activation of cytochrome P-450cam|
|Author(s):||Gerber, Nancy Counts|
|Doctoral Committee Chair(s):||Sligar, Stephen G.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The Cytochrome P-450 class of monoxygenases carry out a wide variety of hydroxylation, epoxidation and heteroatom oxidation reactions. The cytochrome P-450cam enzyme from P. putida has been widely studied as a model for other P-450s due to the availability of a high resolution X-ray crystal structure. In this thesis several questions concerning the activation of P-450cam by substrate, electron transfer and oxygen cleavage are addressed. The identity of the cysteine ligands to the (2Fe-2S) center of P-450cam's electron transport partner, putidaredoxin (Pdx), is determined using a combination of site-directed mutagenesis and EPR spectroscopy. The identity of the rapidly reacting cysteine in P-450cam, which has been used as an attachment site for probes of Pdx-P450cam interaction, is identified. The roles of several amino acids in and around the P-450cam active site are studied in order to better understand their action in substrate access and binding. To help determine the location of the substrate access channel in P-450cam, a novel disulfide bridge and a site for an intraprotein chemical crosslink have been engineered and characterized.
Using site-directed mutagenesis, residues Asp251, Thr252, and Lys178 in P-450cam have been changed in an effort to understand more fully the reaction mechanism of cytochrome P-450cam, particularly as it pertains to the second electron transfer and oxygen bond cleavage steps. The mutant Asp251Asn is characterized in detail in order to determine the origin of the very slow rate of reaction of the protein. The evidence presented in this thesis suggests that the rate limiting step of the Asp251Asn reaction cycle is the scission of the O-O bond, and a mechanism is proposed to explain the effects that the mutations of Asp251, Lys178 and Thr252 have on P-450 catalysis. Based on homology with other P-450s, previous mutagenesis studies and the recently published crystal structure of another P-450, it is believed that the roles for Thr252 and Asp251 proposed in this thesis are common ones in P-450 systems.
|Rights Information:||Copyright 1993 Gerber, Nancy Counts|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9411631|