NADPH-cytochrome P450 enzymes

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

The microsomal fraction consists mainly of vesicles (microsomes) derived from the endoplasmic reticulum (smooth and rough). It contains cytochrome P450 and NADPH/cytochrome P450 reductase (collectively the microsomal monooxygenase system), carboxylesterases, A-esterases, epoxide hydrolases, glucuronyl transferases, and other enzymes that metabolize xenobiotics. The 105,000 g supernatant contains soluble enzymes such as glutathione-5-trans-ferases, sulfotransferases, and certain esterases. The 11,000 g supernatant contains all of the types of enzyme listed earlier. 

Eight human brain tumors contained DT-diaphorase, NADH cytochrome b5 reductase and NADPH cytochrome P450 reductase as assessed by enzyme activity and WB (Rampling et al., 1994). 

Cinnamate 4-hydroxylase (C4H EC 1.14.13.11, also defined as CYP73A [36]) catalyzes the p-hydroxylation of trani-cinnamate to form trani -p-coumarate. The enzyme has a requirement for molecular oxygen and NADPH as well as association with the electron donor NADPH-cytochrome P450 reductase (CPR EC 1.6.2.4) for activity. C4H was the first characterized plant P450 [37, 38]. 

How the NOS isoforms compare to these related dual flavin enzymes is a matter of ongoing investigation. Characterization of the neuronal NOS revealed that it normally exists in its one-electron reduced form and maintains an air-stable, flavin semiquinone radical (Stuehr and Ikeda-Saito, 1992), as seen for NADPH-cytochrome P450 reductase. It is unknown which flavin in NOS contains the odd electron, although precedent argues that it probably resides on... 

Flavin redox states in a dual flavin enzyme. (Left) Single-electron reduction of the isoalloxazine ring generates the semiquinone radical, while reduction by two electrons generates the fully reduced species. (Right) Five possible oxidation levels of a dual flavin enzyme, where the FMN reduction potential is held at a more positive value relative U) FAD. The flavins can theoretically accept a maximum of four electrons obtained from two NADPH. However, in NADPH-cytochrome P450, reductase, full reduction of the flavins is not normally reached when NADPH serves as the reductant. 

Murakami, H., Yabusaki, Y., Sakaki, T., Shibata, M. Ohkawa, H. (1987). A genetically engineered P450 monooxygenase construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase. DNA, 6, 189—97-... 

In the catalytic cycle of CYP, reducing equivalents are transferred from NADPH to CYP by a flavoprotein enzyme known as NADPH-cytochrome P450 reductase. The evidence that this enzyme is involved in CYP monooxygenations was originally derived from the observation that cytochrome c, which can function as an artificial electron acceptor for the enzyme, is an inhibitor of such oxidations. This reductase is an essential component in CYP-catalyzed enzyme systems reconstituted from purified components. Moreover antibodies prepared from purified reductase are inhibitors of microsomal... 

For most drugs, oxidative biotransformation is performed primarily by the mixed-function oxidase enzyme system, which is present predominantly in the smooth endoplasmic reticulum of the liver. This system comprises (1) the enzyme NADPH cytochrome P450 reductase (2) cytochrome P450, a family of heme-containing proteins that catalyze a variety of oxidative and reductive reactions and (3) a phospholipid bilayer that facilitates interaction between the two proteins. Important exceptions to this rule are ethyl alcohol and caffeine, which are oxidatively metabolized by enzymes primarily present in the soluble, cytosolic fraction of the liver. 

Monooxygenation reactions involve the reduction of one atom of molecular oxygen to water and the incorporation of the other oxygen atom into the substrate [Eq. (9.1)]. The electrons involved in the reduction of CYP are transferred from NADPH by another enzyme called NADPH-cytochrome P450 oxidoreductase (CYPOR). 

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