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J. Biol. Chem., Vol. 263, Issue 35, 18842-18849, Dec, 1988
WM Atkins and SG Sligar
The role of the active site hydrogen bond of cytochrome P-450cam has been
studied utilizing a combination of site-directed mutagenesis and substrate
analogues with altered hydrogen bonding capabilities. Cytochrome P-450cam
normally catalyzes the regiospecific hydroxylation of the monoterpene
camphor. The x-ray crystal structure of this soluble bacterial cytochrome
P-450 (Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and
Kraut, J. (1985) J. Biol. Chem. 260, 16122- 16128) indicates a specific
hydrogen bond between tyrosine 96 and the carbonyl moiety of the camphor
substrate. The site-directed mutant in which tyrosine 96 has been changed
to a phenylalanine and the substrate analogues thiocamphor and camphane
have been used to probe this interaction in several aspects of catalysis.
At room temperature, both the mutant enzyme with camphor and the wild type
enzyme with thiocamphor bound result in 59 and 65% high-spin ferric enzyme
as compared to the 95% high spin population obtained with native enzyme and
camphor as substrate. The equilibrium dissociation constant is moderately
increased, from 1.6 microM for the wild type protein to 3.0 and 3.3 microM
for wild type-thiocamphor and mutant-camphor complexes, respectively.
Camphane bound to cytochrome P-450cam exhibits a larger decrease in high
spin fraction (45%) and a correspondingly larger KD (46 microM), suggesting
that the carbonyl moiety of camphor plays an important steric role in
addition to its interaction as a hydrogen bond acceptor. The absolute
regioselectivity of the mutant enzyme, and of the wild type enzyme with
thiocamphor, is lost resulting in production of several hydroxylated
products in addition to the 5-exo-hydroxy isomer. Based on rates of NADH
oxidation, comparison of the substrate specificity for these systems
(kcat/KD) indicates a 5- and 7-fold decrease in specificity for the mutant
enzyme and thiocamphor-wild type complex, respectively. The replacement of
the cytochrome P-450cam active site tyrosine with phenylalanine does not
affect the branching ratio of monooxygenase versus oxidase chemistry or
peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J. Am. Chem.
Soc. 109, 3754-3760).
The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by site-directed mutagenesis
Department of Biochemistry, University of Illinois, Urbana 61801.
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