14. Cytochrome P540 1+2 PDF

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Lecture notes from Biochemistry, Year 2020/21....

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CYTOCHROMES P450 They are a unique family of heme proteins encoded by a gene superfamily with hundreds of members. They are enzymes that catalyse monooxygenation of structurally diverse compounds -

endogenous substrates include cholesterol, steroid hormones, and fatty acids. exogenous substrates include drugs, food additives, pesticides and chemicals that enter the body by ingestion, inhalation, and absorption through the skin.

They also make significant contributions to medicine through inactivation or activation of therapeutic agents, production of steroid hormones, enzyme inhibition or induction that results in drug-drug interactions and adverse effects and opportunities for personalised medicines. 1. STRUCTURE: Cys- ligated heme in the active site.

Cys as a heme ligand allows breaking of O-O bond in O2. His as heme ligand allows reversible binding of O2 so that it can be delivered from lungs to cells throughout body where it is needed as terminal electron acceptor for oxidative phosphorylation and ATP synthesis

2. Cytochromes P450 catalyse monooxygenation Monooxygenation is the incorporation of one of two O atoms from O2 into an organic molecule R3CH. R3CH + O2 + NADPH + H+  R3COH + NADP+ + H2O (R3COH is a monooxygenation) where R3CH is a hydrophobic / lipophilic compound, and, R3COH is less hydrophobic and more hydrophilic than R3CH.

R3CH may be a steroid, fatty acid, drug, or other chemical that has an alkane, alkene, aromatic ring or heterocyclic ring substituent.

3. Cytochrome P450 electron transport systems.

NADP+ takes 2 electrons and a proton to be reduced to NADPH. The ion inside the heme cofactor can only exists as Fe2+ or Fe3+. Fe can only take 1 electron at a time so in order to turn 2 electrons into 1 electron step we need a cofactor: Flavin can take 2 e from NADP, passing them 1 at a time to the heme redox cofactor. Most mammalian cytochromes P450 are found in the endoplasmic reticulum within the hepatocytes, in renal cells and cells of the respiratory tract. These cells are the Class II cytochrome P450.

In right hand heme P450, and next to that a 2nd protein containing flavin. This is the strategy that p450 uses in the endoplasmic reticulum. It needs oxidoreductase

Class I cytochrome P450 enzymes are found in mitochondria. With ferredoxin as the cofactor regulating the one at a time rule for cytochrome heme p450

4. A superfamily with hundreds of members. There are hundreds and hundreds of cytochrome p450. The reason for that is because 1 gene can produce multiple cytochromes p450 through isoforms, although they have slightly different activities.

They have a numeric code to differentiate the functions of the cytochrome p450. The number defines the family where the isoform belongs to, then depending on the percentage they are classified in subfamilies. Through recognition they are given a number and a letter to identify each unique member of the subfamily. In humans, there are 57 cytochromes p450, 7 of them are mitochondrial. 5. Cytochrome p450 for processing endogenous molecules. Cytochromes P450 with high substrate specificity process cholesterol, steroids, prostaglandins, and fatty acids. A series of reactions catalysed by CYP11A1 in the adrenal mitochondria are shown here. e.g. Cholesterol to steroid hormones. CYP11A1 transforms cholesterol into pregnenolone. 1. Hydroxyl on C22 is added 2. Alcohol group added to C 3. Unstable molecule and C-C bond is broken. 4. Molecule is cut into 2. But this is only a step I a huge scale.

It takes different pathways to transform cholesterol into hormones covering different important functions. A lot of cytochromes are involved in these steps and other essential processes.

5. Cytochromes P450 are membrane associated. Their substrates are mainly hydrophobic and soluble in lipid bilayers, where lower regions are involved.

6. Cytochromes P450 processing xenobiotics. Cytochromes P450 that process exogenous molecules, xenobiotics, are much less substrate specific than those for processing endogenous molecules. They probably evolved to allow lipophilic toxins acquired from the environment to be made more water soluble for excretion via kidneys or in bile. Three of families are involved in processing many environmental contaminants, food additives, drugs etc… CYP1, CYP2 and CYP3 (with 23 isoforms). Many different substrates can bind adjacent to the catalytic Cys-ligated heme because of the large and cavernous active site pocket within the structure of these proteins.

Why is the hydroxylation so good to remove the xenobiotic from the body? As it introduces alcohol groups, they become more water soluble and at the end being able to be removed from the body. Many xenobiotics are highly lipophilic, and they will accumulate within cells potentially interfering with cellular function unless they are metabolized to more hydrophilic products and then excreted from the body. Types of Reactions Catalysed by Cytochromes P450

7. Cytochromes p450 and metabolism of therapeutic drugs. Broad substrate specificity of CYPs for exogenous substrates can also result in compounds being processed by multiple CYPs at more than one site. CYP3A4 is present in gastrointestinal tract and liver. It is responsible for the poor bioavailability of many drugs because it hydroxylates them to inactive forms.

8. Quantifying CYP catalytic activity. We measure the cytochrome activities with an UV spectrophotometer. One of the substrates of all cytochrome P450 reactions gives a characteristic colour change when it is converted to the corresponding product.

Spectrophotometric assays of steady-state enzyme activity are powerful tools in quantifying the activities of cytochromes P450 to identify the consequences of polymorphisms and the consequences of drug:drug interactions. 9. Steady-state assay of cytochromes P450.

10. Substrate inhibition of cytochromes p450. Most cytochrome P450 catalysed reactions are well-described by the classical MichaelisMenten description of an enzyme catalysed reaction. However, in some cases substrate inhibition is observed as illustrated below for two reactions catalysed by CYP 3A4.

11. Moderated drug efficacy: Substrate inhibition and drug:drug interactions. Explained by the cavernous pocket adjacent to the catalytic heme cofactor.

Regulation of CYP expression.

People with high levels of CYP2E1 are at higher risk for hepatitis as an adverse reaction to anaesthetic. Alcohol and certain anti-depressants trigger increased levels CYP2E1 in the body. As a consequence, those with chronic alcohol intake and/or taking those drugs are at increased risk of developing hepatitis after surgery. The increased levels of CYP2E1 that occur in response to certain anti-depressants or chronic alcohol are an example of drug induced regulation of cytochrome P450 expression. In other cases, expression is decreased.

CYP Polymorphisms.

Polymorphism is a difference in DNA sequence found at 1% or higher in a population. It is thought to occur in genes of 40% of drug metabolising cytochromes P450. As a consequence, individuals contain unique cytochrome P450 genes or alleles and can exhibit very different rates of metabolising individual drugs. Specific genetic variations are often associated with specific ethnic groups. CYP polymorphisms occur in CYP 2C9 found in human liver where it processes several commonly used drugs such as non-steroidal anti-inflammatory drugs and S-warfarin. Wild-type CYP2C9*1 (shown here) Ile359 and Asp360. Polymorphisms in CYP2C9 include: -

Caucasian variant CYP2C9*3 Leu359 (0.4% of population are homozygous carriers and 15% are heterozygous).

African American variant CYP 2C9*5 - Glu360 (approximately 3% of this population carries the CYP2C9*5 allele). CYP2C9 and Warfarin. CYP2C9 is solely responsible for the metabolism of S-warfarin and its elimination from the body. This drug is orally administered over weeks to inhibit blood coagulation in patients who have suffered heart attack or stroke in order to prevent reoccurrence of clots that may cause another life-threatening episode. Too much drug will cause uncontrolled bleeding and too little has no effect so the level in blood must be tightly maintained in a specific range. For the majority of the population, a 4 to 5 mg dose of warfarin per day is beneficial. However, substitution of Leu359 in CYP2C9 causes a substantial loss of enzymatic activity and this is found in the CYP2C9*3 allelic variant. If a 5 mg dose of warfarin was taken by patient with CYP2C9*3 uncontrollable bleeding could result from a simple cut. In such individuals a warfarin dose of 0.5 to 1 mg per week may be sufficient to maintain blood levels of warfarin at the therapeutically appropriate level. 12. Pharmacogenetics: How genes influence an individual’s response to drugs.

Detailed knowledge of the cytochromes P450 within an individual could lead to personalised prescriptions. The Roche AmpliChip CYP450 Test aims to ‘aid to clinicians in determining therapeutic strategy and treatment dose for therapeutics metabolized by the CYP2D6 or CYP2C19 gene product’.

PART 2 13. Understanding the catalytic cycle of cytochromes P450. Considering the overall transformation catalysed by cytochromes P450 raises a number of questions about the order of events that occur in the catalytic cycle:  Must one substrate bind before the others?  Where are the substrates bound within the active site?  What is the role of the heme cofactor in catalysis?  Are the products released simultaneously or in a defined order? It turns out catalysis by most cytochromes P450 occurs is a precisely ordered sequence of events in order to minimise unwanted side-reactions of the very reactive reduced oxygen species that are intermediates in the reaction pathway. The mechanism of cytochromes P450 has been revealed by numerous experiments designed to explore the role of key biochemical concepts; specifically with regard to reaction kinetics, binding constants and redox chemistry. The first steps in the catalytic cycle have been revealed by UV-visible absorbance spectroscopy. 1) Oxidised cytochrome p450 which is oxidised because the iron inn the heme cofactor is in the Fe III oxidation state. First, the organic substrate binds into the active site pocket with the Fe (III) state. 2) An electron is delivered into the enzyme and it is accepted by the heme iron which now becomes reduced to Fe (II).

3) Once it is reduced, the oxygen can bind to the reduced heme. 4) The subsequence step in catalysis is the binding of an electron and a proton that triggers now with 2 electrons in the enzyme the release of O2- and a water molecule is released. 5) Now, with a single oxygen atom bound to Fe heme group, which can be inserted into the carbon hydrogen carbon bond. The monooxygenation occurs of the organicsubstrate. 6) The final step of the catalytic cycle is when the monoxygenated proton is released and the enzyme returns to the starting state. 14. Colour and UV-vis spectroscopy. Because of the presence of the heme cofactor, it has a red colour when Fe (III) is purified, so it gives absorbance when spectroscopy is done with a maximum absorbance at 410-420 nm. In the peak of absorbance, it is when it is oxidised and where there is no absorbance of the visible light, it will determine the colour. 15. UV-vis Spectra: oxidised and reduced cytochrome p450. The protein was exposed to the chemical reductant, sodium dithionite Eo’ -500 mV, to generate the sample producing the spectrum of reduced protein above. The spectrum of the heme cofactor has changed and we can guess that heme cofactor is a redox cofactor.

Spectroscopy has identified the cofactor in cytochrome P450. Changes in the spectrum on addition of a chemical reductant, sodium dithionite, indicate the cofactor is redox active. Spectroscopy can also be used to define the parameters that quantify the redox activity. 16. Redox chemistry, the Nernst equation and reducing power.

The most positive the reductio potential is, the greater the affinity the oxidised molecule has for the electrons. O2 is the strongest oxidising agent or oxidant. NADPH is the strongest reducing agent or reductant.

17. Substrate Binding Alters the Reduction Potential of P450 Aliquots of the chemical reductant, sodium dithionite Eo’ -500 mV, used to reduce the enzyme. In one experiment no organic substrate is present (black line on right), in one experiment an organic substrate is present (orange broken line). Conclusion: In the presence of the reduction potential of cytochrome p450 becomes significantly more positive, Binding of the organic substrate causes water molecules to change their positionin the active site and this changes the environment and reduction potential of the heme. R3CH Binding to P450 Allows Heme Reduction by NADPH

NADPH is not enough reducing agent to pass the ion in the cytochrome p450 through ion transport in a thermodynamically way, but if it comes more positive with organic substrate, it changes the thermodynamic profile cause now NADPH is enough reducing agent to reduce the heme in the protein when the organic substrate is bound.

R3CH binding to P450 Fe (III) raises Eo’ of the P450 Fe (III)/(II) couple such that it is now less negative than for NADP+/NADPH. NADPH can now reduce the P450 and electron transfer occurs. The

redox analysis allows us the understand the first two steps in the reaction cycle: the organic substrate siting in the pocket, allowing electrons to come into the active site and reduce the Fe III into Fe II. O2 binds only after organic substrate and an e- are present in the active site. This ensures no reactive oxygen species can be formed. The ordered reaction mechanism minimises the opportunity for cytochromes P450 to reduce O2 prior to binding the organic substrate. This minimises the production of reactive oxygen species, shown in the blue below, that are harmful to cells. In Cytochromes P450 when O2 binds the reduced heme the organic substrate is present, and this determines the reaction products....