کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1925256 | 1536354 | 2014 | 8 صفحه PDF | دانلود رایگان |

• Absorbance spectroscopy revealed two amlodipine binding events in solution with P450 2B6 and 2B4.
• The results are in agreement with recently solved X-ray structures of the amlodipine complexes.
• The function of access channel residues was determined by site-directed mutagenesis and steady-state kinetics.
• R73 in P450 2B6 and R73, V216, L219, and F220 in 2B4 are important for catalytic efficiency.
Recent X-ray crystal structures of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with amlodipine showed two bound ligand molecules, one in the active site and one in the substrate access channel. Based on the X-ray crystal structures, we investigated the interactions of P450 2B4 and 2B6 with amlodipine using absorbance spectroscopy, and determined the steady-state kinetics of 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin oxidation by some access channel mutants to evaluate the functional role of these residues in substrate turnover. The results of absorbance titrations are consistent with a simple mechanism with two parallel binding events that result in the formation of the enzyme complex with two molecules of amlodipine. Using this model we were able to resolve two separate ligand-binding events, which are characterized by two distinct KD values in each enzyme. The access channel mutants R73K in P450 2B6 and R73K, V216W, L219W, and F220W in P450 2B4 showed a significant decrease in kcat/KM with the both substrates. Overall, the results suggest that P450 2B4 and 2B6 form an enzyme complex with two molecules of amlodipine in solution, and R73, V216, L219 and F220 in P450 2B4 may play an important role in substrate metabolism.
Journal: Archives of Biochemistry and Biophysics - Volume 545, 1 March 2014, Pages 100–107