Format

Send to

Choose Destination
J Biol Chem. 2009 Sep 18;284(38):25678-86. doi: 10.1074/jbc.M109.032748. Epub 2009 Jul 15.

Effect of conformational dynamics on substrate recognition and specificity as probed by the introduction of a de novo disulfide bond into cytochrome P450 2B1.

Author information

1
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA.

Abstract

The conformational dynamics of cytochrome P450 2B1 (CYP2B1) were investigated through the introduction of a disulfide bond to link the I- and K-helices by generation of a double Cys variant, Y309C/S360C. The consequences of the disulfide bonding were examined both experimentally and in silico by molecular dynamics simulations. Under high hydrostatic pressures, the partial inactivation volume for the Y309C/S360C variant was determined to be -21 cm3mol(-1), which is more than twice as much as those of the wild type (WT) and single Cys variants (Y309C, S360C). This result indicates that the engineered disulfide bond has substantially reduced the protein plasticity of the Y309C/S360C variant. Under steady-state turnover conditions, the S360C variant catalyzed the N-demethylation of benzphetamine and O-deethylation of 7-ethoxy-trifluoromethylcoumarin as the WT did, whereas the Y309C variant retained only 39% of the N-demethylation activity and 66% of the O-deethylation activity compared with the WT. Interestingly, the Y309C/S360C variant restored the N-demethylation activity to the same level as that of the WT but decreased the O-deethylation activity to only 19% of the WT. Furthermore, the Y309C/S360C variant showed increased substrate specificity for testosterone over androstenedione. Molecular dynamics simulations revealed that the engineered disulfide bond altered substrate access channels. Taken together, these results suggest that protein dynamics play an important role in regulating substrate entry and recognition.

PMID:
19605359
PMCID:
PMC2757969
DOI:
10.1074/jbc.M109.032748
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center