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NMR-Derived Models of Amidopyrine and Its Metabolites in Complexes with Rabbit Cytochrome P450 2B4 Reveal a Structural Mechanism of Sequential N-Dealkylation

Roberts, Arthur G.; Sjögren, Sara LU ; Fomina, Nadezda; Vu, Kathy T.; Almutairi, Adah and Halpert, James R. (2011) In Biochemistry 50(12). p.2123-2134
Abstract
To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T-1) relaxation NMR. Results of STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme's active site. The model-dependent relaxation rates (R-M) determined from T-1 relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine... (More)
To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T-1) relaxation NMR. Results of STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme's active site. The model-dependent relaxation rates (R-M) determined from T-1 relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine the orientation(s) of AP and its metabolites within the P450 2B4 active site, we used distances calculated from the relaxation rates to constrain the metabolites to the X-ray crystal structure of P450 2B4. Simulated annealing of the complex revealed that the metabolites do indeed occupy similar hydrophobic pockets within the active site, while the N-linked methyls are free to rotate between two binding modes. From these bound structures, a model of N-demethylation in which the N-linked methyl functional groups rotate between catalytic and noncatalytic positions was developed. This study is the first to provide a structural model of a drug and its metabolites complexed to a c-ytochrome P450 based on NMR and to provide a structural mechanism for how a drug can undergo sequential oxidations without unbinding. The rotation of the amide functional group might represent a common structural mechanism for N-dealkylation reactions for other drugs such as the local anesthetic lidocaine. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
50
issue
12
pages
2123 - 2134
publisher
The American Chemical Society
external identifiers
  • wos:000288573500016
  • scopus:79952931175
ISSN
0006-2960
DOI
10.1021/bi101797v
language
English
LU publication?
yes
id
d2a5c0c8-edc8-4303-9910-bc0f937626e4 (old id 1926164)
date added to LUP
2011-05-10 13:48:18
date last changed
2017-04-09 03:15:53
@article{d2a5c0c8-edc8-4303-9910-bc0f937626e4,
  abstract     = {To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T-1) relaxation NMR. Results of STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme's active site. The model-dependent relaxation rates (R-M) determined from T-1 relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine the orientation(s) of AP and its metabolites within the P450 2B4 active site, we used distances calculated from the relaxation rates to constrain the metabolites to the X-ray crystal structure of P450 2B4. Simulated annealing of the complex revealed that the metabolites do indeed occupy similar hydrophobic pockets within the active site, while the N-linked methyls are free to rotate between two binding modes. From these bound structures, a model of N-demethylation in which the N-linked methyl functional groups rotate between catalytic and noncatalytic positions was developed. This study is the first to provide a structural model of a drug and its metabolites complexed to a c-ytochrome P450 based on NMR and to provide a structural mechanism for how a drug can undergo sequential oxidations without unbinding. The rotation of the amide functional group might represent a common structural mechanism for N-dealkylation reactions for other drugs such as the local anesthetic lidocaine.},
  author       = {Roberts, Arthur G. and Sjögren, Sara and Fomina, Nadezda and Vu, Kathy T. and Almutairi, Adah and Halpert, James R.},
  issn         = {0006-2960},
  language     = {eng},
  number       = {12},
  pages        = {2123--2134},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {NMR-Derived Models of Amidopyrine and Its Metabolites in Complexes with Rabbit Cytochrome P450 2B4 Reveal a Structural Mechanism of Sequential N-Dealkylation},
  url          = {http://dx.doi.org/10.1021/bi101797v},
  volume       = {50},
  year         = {2011},
}