BIOCHEMISTRY - DR. JAKUBOWSKI
Learning Goals/Objectives for Chapter 8B: After class and this reading, students will be able to
An examples of monooxygenases are the hydroxylases which hydroxylate amino acids like Trp and Tyr to form 5-hydroxytrytophan and 3-4-dihydroxyphenylalanine or dopa, respectively. These latter two substances can be decarboxylated using PLP-dependent enzymes to form the neurotransmitters 5 hydroxytryptamine (5HT or serotonin) and dopamine. The latter can be hydroxylated again to form norepinhephrine, and subsequently methylated to form epinephrine. LSD and amphetamine are anaologs of serotonin and dopamine, respectively.
Figure: DIAGRAM: TRP AND TYR CHEMISTRY
Since these monooxygenases use dioxygen, you might expect that the enzymes would use the motifs described in the previous section to facilitate its reaction with dioxygen. In fact, the enzyme contains a metal ion (Fe2+) bound to a heme in the protein. In addition, the reduction products of dioxygen that are eventually used to hydroxylate the substrate stay bound to the enzyme.
An important class of monoxygenases are called cytochromes P450's. They represent a class of similar enzymes that each contain a heme. Instead of reversibly carrying dioxygen as does the heme in myoglobin and hemoglobin, the P450 heme activates dioxgyen for hydroxylation reactions involving aromatic, nonpolar substrates. Hydroxylation of these substrates increases their solubility which facilitates their elimination from the body. Epoxide intermediates or products are often produced, which can open up through nucleophilic attack using an alcohol (sugar derivative) or an amine (such as in nucleotide bases in DNA) to form large adducts. Hence cytochrome P450 can actually activate aromatic substrates to become carcinogens.
The cytochrome P450s family of genes/proteins are inducible on exposure to nonpolar aromatic molecules such as dioxin. These nonpolar molecules can enter the cytoplasm where they bind to the arylhydrocarbon receptor (AhR) which is bound to a heat shock protein, Hsp90. Upon binding of dioxin, TCDD, for example, the AhR.TCCD complex dissociates from Hsp90, and migrates to the nucleus where it binds a protein called Amt. The AhR-Amt complex serves as an enhancer/transcription factor, facilitating the transcription of the cytochrome P450 genes.
Figure: Cytochrome P450s inducible on exposure to nonpolar aromatic molecules such as dioxin.
Dioxin has been shown to affect estrogen-mediated activities. Estrogens, small hydrophobic hormones derived from cholesterol, enter cell and bind to cytoplasmic estrogen receptors, which then dimerize and bind to the estrogen response element (ERE), initiating transcription. Recently the crystal structure of the ligand binding domain of the estrogen receptor, bound to tamoxifen, was reported. Tamoxifen, a drug derived from the yew plant, blocks the biological effects of the estrogen receptor. Although it binds to the estrogen receptor, it doesn't elicit the same conformational changes in the protein, which prevents the bound receptor from binding to the estrogen response element and recruiting other proteins needed for estrogen-dependent gene transcription. It is used in chemotherapy and prevention of estrogen-dependent breast cancer cells.
How does dioxin interfer with estrogen signaling? As mentioned above, dioxin binds to the arylhydrocarbon receptor (AhR). This complex can then bind to Arnt - arylhydrocarbon nuclear transporter (a chaperone). (Note: unclear about AMT, Arnt relationship. Will fix soon). This ternary complex can then bind to the xenobiotic response element (XRE). Ahr and ARnt contain a basic helix-loop-helix motif which mediate their interaction with DNA. Upon binding of the complex, detoxification genes are activated. Ohtake et al. found that the dioxin-Ahr-Arnt complex can bind to the estrogen receptor, which can then lead to activation of the ERE in the absence of estrogen. However, if estrogen is present, inhibition of gene expression from ERE is observed. Dioxins can be potent disregulators of estrogen-induced gene expression. Such changes in esterogen activity could help to explain the pro- and inhibitory effects of dioxin on estrogen-mediated cellular responses and possible effects of dioxin on the immune system and on cancer development.
EPA Dioxin Reassessment: Risk Characterization - Dose Response
EPA Dioxin Reassessment: Risk Characterization - Mechanisms
Recently, "snapshots" of P450cam, the cytochrome P450 that hydroxylates camphor, have been taken in various stages of catalysis. This enzyme is "the biological equivalent of a blowtorch: P450 enzymes catalyze the stereospecific hydroxylation of nonactivated hydrocarbons at physiological temperature - a reaction that, uncatalyzed, requires extremely high temperatures to proceed, even nonspecifically". Crystal structures of normal intermediates (such as the dioxygen-bound intermediate, could not be determined with traditional techniques since the complex had a half-life of 10 minutes at 4oC. This problem was solved by using crystals frozen at -185oC, and by using short wavelength X-rays, which did not cause the reaction to be driven forward. Short-time X-ray data was collected, then substrate added to push the reaction to the next intermediate, before new structural data was obtained.
Figure: Reaction Pathyway of P450cam
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