The roles of active site hydrogen bonding in cytochrome P-450(cam) as revealed by site-directed mutagenesis

W. M. Atkins, S. G. Sligar

Research output: Contribution to journalArticle

Abstract

The role of the active site hydrogen bond of cytochrome P-450(cam) has been studied utilizing a combination of site-directed mutagenesis and substrate analogues with altered hydrogen bonding capabilities. Cytochrome P-450(cam) 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 μM for the wild type protein to 3.0 and 3.3 μM for wild type-thiocamphor and mutant-camphor complexes, respectively. Camphane bound to cytochrome P-450(cam) exhibits a larger decrease in high spin fraction (45%) and a correspondingly larger K(D) (46 μM), 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 (κ(cat)/K(D)) 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-450(cam) active site tyrosine with phenylalanine does not affect the branching ration of monooxygenase versus oxidase chemistry or peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J.Am. Chem. Soc. 109, 3754-3760).

Original languageEnglish (US)
Pages (from-to)18842-18849
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number35
StatePublished - Dec 1 1988

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Camphor 5-Monooxygenase
Mutagenesis
Camphor
Hydrogen Bonding
Site-Directed Mutagenesis
Catalytic Domain
Hydrogen bonds
Enzymes
Substrates
Tyrosine
Hydrogen
Phenylalanine
Bacterial Structures
Cytochrome P-450 CYP2E1
Regioselectivity
Hydroxylation
Monoterpenes
Substrate Specificity
Mixed Function Oxygenases
Catalysis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The roles of active site hydrogen bonding in cytochrome P-450(cam) as revealed by site-directed mutagenesis. / Atkins, W. M.; Sligar, S. G.

In: Journal of Biological Chemistry, Vol. 263, No. 35, 01.12.1988, p. 18842-18849.

Research output: Contribution to journalArticle

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abstract = "The role of the active site hydrogen bond of cytochrome P-450(cam) has been studied utilizing a combination of site-directed mutagenesis and substrate analogues with altered hydrogen bonding capabilities. Cytochrome P-450(cam) 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 μM for the wild type protein to 3.0 and 3.3 μM for wild type-thiocamphor and mutant-camphor complexes, respectively. Camphane bound to cytochrome P-450(cam) exhibits a larger decrease in high spin fraction (45{\%}) and a correspondingly larger K(D) (46 μM), 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 (κ(cat)/K(D)) 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-450(cam) active site tyrosine with phenylalanine does not affect the branching ration of monooxygenase versus oxidase chemistry or peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J.Am. Chem. Soc. 109, 3754-3760).",
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N2 - The role of the active site hydrogen bond of cytochrome P-450(cam) has been studied utilizing a combination of site-directed mutagenesis and substrate analogues with altered hydrogen bonding capabilities. Cytochrome P-450(cam) 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 μM for the wild type protein to 3.0 and 3.3 μM for wild type-thiocamphor and mutant-camphor complexes, respectively. Camphane bound to cytochrome P-450(cam) exhibits a larger decrease in high spin fraction (45%) and a correspondingly larger K(D) (46 μM), 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 (κ(cat)/K(D)) 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-450(cam) active site tyrosine with phenylalanine does not affect the branching ration of monooxygenase versus oxidase chemistry or peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J.Am. Chem. Soc. 109, 3754-3760).

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