Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids

Area of Science:

• Biochemistry
• Membrane Biology
• Pharmacology

Background:

• Cytochrome P-450 enzymes are crucial for drug metabolism.
• Understanding their membrane environment is key to their function.
• Previous studies suggested membrane integration but lacked direct evidence.

Purpose of the Study:

• To investigate the localization of cytochrome P-450 within phospholipid bilayers.
• To determine if the substrate binding site is accessible from the membrane interior or exterior.

Main Methods:

• Incorporation of purified rabbit cytochrome P-450 into dimyristoylphosphatidylcholine liposomes.
• Spectroscopic analysis of benzphetamine binding to liposome-bound and microsomal-bound cytochrome P-450 at varying temperatures.
• Analysis of van't Hoff plots to identify phase transition effects on binding affinity.

Main Results:

• A distinct break in the van't Hoff plot for benzphetamine binding to liposome-bound cytochrome P-450 occurred at the lipid phase transition temperature.
• No such break was observed for benzphetamine binding to cytochrome P-450 in native microsomes.
• This indicates a difference in the microenvironment of the enzyme in liposomes versus microsomes.

Conclusions:

• The substrate binding site of cytochrome P-450, when reconstituted into liposomes, is embedded within the hydrophobic core of the phospholipid bilayer.
• This suggests that the enzyme's active site is not freely accessible from the aqueous phase in a membrane-like environment.
• The findings provide insights into the membrane topology and substrate access mechanisms of cytochrome P-450 enzymes.