Oxygen-18 tracer studies of enzyme reactions with radical/cation diagnostic probes

Luke A. Moe; Brian G. Fox

doi:10.1016/j.bbrc.2005.08.217

Oxygen-18 tracer studies of enzyme reactions with radical/cation diagnostic probes

Luke A. Moe, Brian G. Fox

Research output: Contribution to journal › Review article › peer-review

4 Scopus citations

Abstract

This review considers reactions of enzymes with the cyclopropanoid radical/cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane as elaborated by the use of ¹⁸O₂ and ¹⁸OH₂ to trace the origin of O-atoms incorporated during catalysis. The reactions of soluble and integral membrane diiron enzymes are summarized and compared to results obtained from cytochrome P450 studies. Norcarane proved to be an excellent substrate for the diiron enzyme toluene 4-monooxygenase and its engineered isoforms, with k_cat and coupling between NADH utilization and total hydroxylated products comparable to that determined for toluene, the natural substrate. Results obtained with toluene 4-monooxygenase show that the un-rearranged and radical-rearranged alcohol products have a high percentage of O-atom incorporation (>80-95%) from O ₂, while the cation-derived ring-expansion products have O-atom incorporation primarily derived from solvent water. Mechanistic possibilities accounting for this difference are discussed.

Original language	English
Pages (from-to)	240-249
Number of pages	10
Journal	Biochemical and Biophysical Research Communications
Volume	338
Issue number	1
DOIs	https://doi.org/10.1016/j.bbrc.2005.08.217
State	Published - Dec 9 2005

Bibliographical note

Funding Information:
This work was supported by National Science Foundation Grant MCB-0316232 to B.G.F. The authors acknowledge many useful discussions with our patient collaborators John T. Groves Zhengbo Hu, Dayi Deng (Princeton University) and Rachel N. Austin (Bates College) while the oxygen-18 experiments progressed.

Keywords

Catalysis
Diiron enzyme
Hydroxylation mechanism
Norcarane
Oxygen-18
Radical clock

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.bbrc.2005.08.217

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title = "Oxygen-18 tracer studies of enzyme reactions with radical/cation diagnostic probes",

abstract = "This review considers reactions of enzymes with the cyclopropanoid radical/cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane as elaborated by the use of 18O2 and 18OH2 to trace the origin of O-atoms incorporated during catalysis. The reactions of soluble and integral membrane diiron enzymes are summarized and compared to results obtained from cytochrome P450 studies. Norcarane proved to be an excellent substrate for the diiron enzyme toluene 4-monooxygenase and its engineered isoforms, with kcat and coupling between NADH utilization and total hydroxylated products comparable to that determined for toluene, the natural substrate. Results obtained with toluene 4-monooxygenase show that the un-rearranged and radical-rearranged alcohol products have a high percentage of O-atom incorporation (>80-95%) from O 2, while the cation-derived ring-expansion products have O-atom incorporation primarily derived from solvent water. Mechanistic possibilities accounting for this difference are discussed.",

keywords = "Catalysis, Diiron enzyme, Hydroxylation mechanism, Norcarane, Oxygen-18, Radical clock",

author = "Moe, {Luke A.} and Fox, {Brian G.}",

note = "Funding Information: This work was supported by National Science Foundation Grant MCB-0316232 to B.G.F. The authors acknowledge many useful discussions with our patient collaborators John T. Groves Zhengbo Hu, Dayi Deng (Princeton University) and Rachel N. Austin (Bates College) while the oxygen-18 experiments progressed.",

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AU - Moe, Luke A.

AU - Fox, Brian G.

N1 - Funding Information: This work was supported by National Science Foundation Grant MCB-0316232 to B.G.F. The authors acknowledge many useful discussions with our patient collaborators John T. Groves Zhengbo Hu, Dayi Deng (Princeton University) and Rachel N. Austin (Bates College) while the oxygen-18 experiments progressed.

PY - 2005/12/9

Y1 - 2005/12/9

N2 - This review considers reactions of enzymes with the cyclopropanoid radical/cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane as elaborated by the use of 18O2 and 18OH2 to trace the origin of O-atoms incorporated during catalysis. The reactions of soluble and integral membrane diiron enzymes are summarized and compared to results obtained from cytochrome P450 studies. Norcarane proved to be an excellent substrate for the diiron enzyme toluene 4-monooxygenase and its engineered isoforms, with kcat and coupling between NADH utilization and total hydroxylated products comparable to that determined for toluene, the natural substrate. Results obtained with toluene 4-monooxygenase show that the un-rearranged and radical-rearranged alcohol products have a high percentage of O-atom incorporation (>80-95%) from O 2, while the cation-derived ring-expansion products have O-atom incorporation primarily derived from solvent water. Mechanistic possibilities accounting for this difference are discussed.

AB - This review considers reactions of enzymes with the cyclopropanoid radical/cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane as elaborated by the use of 18O2 and 18OH2 to trace the origin of O-atoms incorporated during catalysis. The reactions of soluble and integral membrane diiron enzymes are summarized and compared to results obtained from cytochrome P450 studies. Norcarane proved to be an excellent substrate for the diiron enzyme toluene 4-monooxygenase and its engineered isoforms, with kcat and coupling between NADH utilization and total hydroxylated products comparable to that determined for toluene, the natural substrate. Results obtained with toluene 4-monooxygenase show that the un-rearranged and radical-rearranged alcohol products have a high percentage of O-atom incorporation (>80-95%) from O 2, while the cation-derived ring-expansion products have O-atom incorporation primarily derived from solvent water. Mechanistic possibilities accounting for this difference are discussed.

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KW - Diiron enzyme

KW - Hydroxylation mechanism

KW - Norcarane

KW - Oxygen-18

KW - Radical clock

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Oxygen-18 tracer studies of enzyme reactions with radical/cation diagnostic probes

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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