Abstract
Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.
Highlights
Cytochrome P450 belongs to a broad spectrum enzymatic systems responsible for detoxifying endogenous substrates, such as, steroids and hormones, as well as exogenous compounds, including drugs and lipophilic hydrocarbons [1]
Phospholipid has been used to reconstitute the enzymatic activity of different purified P450 proteins, including P4501A and P4503A [32,33,34], but an intriguing question remains: How does increased phospholipid influence enzymatic activity of P4501A? Based upon the light of research of Agarwal et al, it can be conceivable that phospholipid may alter the structural conformation of purified protein which in turn augments the enzymatic activity
Polycyclic aromatic hydrocarbons (PAH) pose a serious threat to the environment for their multipotent toxicities on the biosphere [35]. These compounds are reportedly implicated in altered regulation of cytochrome P4501A (CYP1A) expression that is responsible for the detoxification of lipophilic hydrocarbons in the liver of aquatic vertebrates [1,36,37]
Summary
Cytochrome P450 belongs to a broad spectrum enzymatic systems responsible for detoxifying endogenous substrates, such as, steroids and hormones, as well as exogenous compounds, including drugs and lipophilic hydrocarbons [1]. Among all P450s, cytochrome P4501A (CYP1A) is a family of protein that is reportedly involved in degrading the lipophilic substrate known as polycyclic aromatic hydrocarbons [2]. The ability of phospholipid in modulating an enzymatic structural conformation was demonstrated by many workers [4,6,7,8,9,10,11,12]. How structural conformation was intricately involved in regulating the enzymatic activity had elegantly demonstrated by several other authors [13,14,15,16,17]. The conformational studies on P450s from fish with CD has not yet been reported, it is immensely important as the simplified structurefunction relationship in this lower vertebrate may unveil the biochemical and biophysical behavior of this protein during toxic stress in physiological microenvironment
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