The Active Site Structure and the Substrate Specificity of Cytochrome P-450(cam)

191 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.Cytochrome P-450$\sb{\rm cam}$, from Pseudomonas putida, catalyzes the regio- and stereospecific hydroxylation of the monoterpene d-camphor to afford 5-exo-hydroxycamphor as the sole product. The roles of specific active site structural features in dictating this substrate specificity have been determined with the use of site-directed mutagenesis and specifically modified substrate analogues. In particular, Val-295 forms a complementary fit with the 8,9-gem-dimethyl moiety of camphor and the 10-methyl group of camphor is accommodated by a hydrophobic cleft formed by Leu-244, Phe-98, and Val-247. The site-directed mutant Y96F and the substrate analogue thiocamphor, which perturb the active site hydrogen bond, indicate that the major contribution of this bond is in the maintenance of substrate dependent spin state regulation. Both of these hydrogen bond probes afford a mixed spin system with a decrease in the maximal high spin species obtained.The substrate analogue 1-methyl-norcamphor indicates that reduction of complementarity between Val-295 and the camphor gem-dimethyl group is essential for all aspects of substrate specificity, including tight substrate binding, spin state regulation, regiospecificity of hydroxylation, and efficient coupling of NADH consumption with formation of hydroxylated products. The substrate analogue norcamphor, which lacks all of the methyl groups present on camphor, is processed by P-450$\sb{\rm cam}$ to afford 3-exo-, 6-exo-, and 5-exo-hydroxynorcamphor, along with hydrogen peroxide. In addition, stoichiometric analysis indicates that all of the NADH consumed cannot be accounted for by hydroxynorcamphor and hydrogen peroxide production, and the ratio of excess NADH consumed to excess oxygen consumed is 2.1-2.1. The kinetic deuterium isotope effects on hydrogen peroxide formation, oxygen consumption, product formation, and NADH consumption have been determined and a disparity in their magnitudes is evident. The kinetic and stoichiometric deuterium isotope effects are explained by a model.Utilizing norcamphor as a substrate skeleton, the substrate specificity of P-450$\sb{\rm cam}$ can be reconstructed by addition of hydrophobic groups to the active site complex, by substrate modification or mutagenesis of hydrophobic residues. Incremental addition of methyl groups in this manner affords substrate/enzyme complexes with increased regiospecificity for hydroxylation at the 5-position and increased coupling to afford higher yields of hydroxylated products. (Abstract shortened with permission of author.)U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD