number Done by Corrected by Doctor Nayef Karadsheh

Transcription

1 number 17 Done by Abdulrahman Alhanbali Corrected by Lara Abdallat Doctor Nayef Karadsheh 1 P a g e

2 Pentose Phosphate Pathway (PPP) Or Hexose Monophosphate Shunt In this lecture We will talk about the pentose phosphate pathway (PPP). we will explain the pathway in details, its functions, and how does it affect the ROS. The pentose phosphate pathway is a pathway where hexoses get converted to pentoses and vice versa. Functions of the pentose phosphate pathway: 1- Production of NADPH : this the most important function Of this pathway. NADPH is very important because it is a biochemical reductant ( source of the energy rich electrons) which can be used for : A- Reductive biosynthesis like in : 1- fatty acid synthesis in fat producing tissues (e.g.:liver/ adipose tissue /mammary glands during lactating). 2- Biosynthesis of steroids (e.g.: cholesterol in the liver and some steroid hormones in the testes, placenta, ovaries and adrenal cortex). B- Maintaining the level of major reducing anti-oxidant compound - Glutathione- in its reduced form (will be discussed later on). 2- metabolism of pentoses (5-carbon sugars) ; if the body needs pentose sugars (for example: ribose-5-phosphate to synthesize nucleic acids) it make them from hexose sugars through this pathway and vise versa; if it needs hexoses from ingested pentoses it uses this pathway also. Pathway:- Consists of two parts: 1- irreversible oxidative part. 2- Reversible nonoxidative part. 2 P a g e

3 1- Irreversible oxidative part : This part consists of two steps, catalyzed by three different enzymes and both of them yield NADPH. 1 st step: (2 reactions integrated in a single step) This step is important because it is the rate limiting step,, Glucose 6- Phosphate is oxidized by the enzyme Glucose 6-Phosphate Dehydrogenase (G6PD), which means the aldehyde group in glucose 6-P is oxidized to carboxyl group,resulting in 6-Phosphogluconolactone,at the same time, NADP+ is reduced to NADPH. Then 6-Phosphoglucolactone is Hydrolysed by the enzyme, 6- Phosphoglucolactone Hydrolase into, 6-phosphogluconate and this step doesn't produce NADPH. 3 P a g e

4 Glucose-6-Phosphate Dehydrogenase is the most common intracellular enzyme to have a disease-producing abnormality. The enzyme G6DP is highly regulated -because as we mentioned this the rate limiting step-. It is inhibited by excess NADPH "feedback inhibition". 2 nd step : The oxidative decarboxylation of the product, 6-phosphogluconate, is catalyzed by 6-phosphogluconate dehydrogenase, yielding CO2(from carbon 1 of glucose), NADPH and the first pentose in this pathway; ribulose- 5-phosphate. ( Ribulose is the isomer of Ribose and it is a ketose as the name indicates ul ) To summarize oxidative reactions : Glucose 6 Phosphate +2 NADP+ Ribulose 5-Phosphate + CO2 + 2NADPH - So if we have six Glucose 6-P molecules for example, how many NADPH molecule are they going to give? The answer is 12. This portion of the pathway is particularly important in the liver, lactating mammary glands, and adipose tissue, which are active in the NADPH-dependent biosynthesis of fatty acids; in the testes ovaries, placenta, and adrenal cortex, which are active in the NADPH-dependent biosynthesis of steroid hormones; and in red blood cells (RBCs), which require NADPH to keep glutathione reduced. 2- Reversible nonoxidative part: In this part pentose will be converted back to hexose through multiple reversible steps (that s why this pathway is also called hexose monophosphate shunt pathway). In this part three pentoses are converted to two hexoses and a single triose. The path depends on the cells needs, whether it needs ribose 5- phosphate for the DNA or RNA synthesis, or the need of NADPH. Seven simple rules you need to know about nonoxidatvie reactions: 4 P a g e

5 1- Nonoxidative reactions in the (PPP) are ONLY rearrangements of sugars, which means that there won t be any CO2 release or ATP production or oxidation or reduction. 2- Three pentose phosphates will eventually give two hexose phosphates and one triose phosphate. -Three pentoses =15 carbon atom -Two hexoses +one triose =15 carbon atom Same number of carbon atoms; that s why we said it s only rearrangement of sugars. 3 pentose phosphate 2 hexose phosphate +1 triose phosphate 3- They are REVERSIBLE reactions. 4- Transfer of 2 or 3 carbon fragment from sugar to sugar. 5- Transketolase transfer 2 carbons and Transaldolase transfer 3 carbons. 6- Ketose + Aldose Ketose + Aldose.. and this reaction is reversible as we said 7- The transfer always occurs from Ketose to Aldose (the donor is the Ketose and the acceptor is the aldose). 5 P a g e

6 1 st step: ribulose can either undergo epimerization yielding xylulose 5- phosphate by phosphopentose Epimerase Or isomerization yielding the aldose ribose 5-phosphate by ribose 5-Phosphoisomerase (the latter is very important For nucleic acid synthesis). 2 nd step: catalyzed by the enzyme transketolase; a two-carbon segment is transferred from xylulose 5-phosphate to ribose 5-phosphate leaving us a seven carbons sugar called sedoheptulose 7-phosphate and the three carbon sugar glyceraldehyde 3-phosphate. Transketolase requires the cofactor thiamine pyrophosphate TPP (vitamin b1) to complete this reaction. *reminder : TPP is needed by : 1-transketolases 2-pyruvate dehydrogenase 3- a-ketoglutarate dehydrogenase 4-(we didn t take this yet) branched chain a-keto acid dehydrogenase 3 rd step: the enzyme transaldolase transfers a three-carbon segment from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate to make erythrose 4-phosphate and the first hexose in the nonoxidative 6 P a g e

7 part; fructose 6-phosphate. The latter can continue in glycolysis or the other reactions we took previously. 4 th step: once again, transketolase moves a two-carbon segment from another xylulose 5-phosphate but this time to erythrose 4-phosphate and make the second fructose 6-phosphate of this pathway and a glyceraldehyde 3-phosphate which can also continue in glycolysis or other reaction. Each 3 hexoses (or pentoses) enter this pathway yield 2 fructose 6- phosphates and a glyceraldehyde 3-phosphate in addition to (in hexoses case only) 6 NADPH and 3 molecules of CO2. the NET nonoxidative reaction is: 3 Ribulose 5-Phosphate 2 fructose 6-phosphate +Glyceraldehyde 3- Phosphate A good article about PPP: NADPH vs NADH: Structurally, the only difference between NADPHand NADH is the phosphate group on the second carbon of ribose in NADPH in comparison to a H atom in the NADH. But functionally, there are some significant differences: A) Each one is specific for certain enzymes; in other words,nadph enzymes don t use NADH and vise versa. B) They differ with their reduced state to oxidized state ratio; while NADH present mainly in the oxidized state (NAD+) because of its oxidative role, the predominant state of NADPH is the reduced state and that s because it s used in reductive biosynthesis and in harvesting harmful oxidative products.(in hepatocytes for example, 7 P a g e

8 the NADP+/ NADPH ratio is about 1/10 while the NAD+/NADH ratio is 1000/1). C) NADH uses the high energy electrons for energy production, whereas NADPH uses high energy electrons for reductive biosynthetic pathways and for oxidants' reduction. Uses of NADPH: As mentioned before NADPH can be used for: A) reductive biosynthesis like steroids and fatty acids synthesis (this will be discussed later in the lipid metabolism chapter). B) Reduction of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide ions and the deadly hydroxyl free radicle. These are normal products of our aerobic metabolism; but they are formed in amounts that could be handled by our Antioxidants mechanisms. However, if the body is exposed to chemicals, smoking, radiation etc. their level will increase above the Antioxidative stress mechanisms capacity and they can damage proteins, lipids and most important the nucleic acids which will cause disorders among the body systems, they can cause cancer, inflammatory disease and aging. C) Cytochromes P450: NADPH is also required by a family of monooxygenases called cytochromes P450. D) Phagocytosis by the white blood cells. E) Nitric oxide synthesis. 8 P a g e

9 Anti-oxidant enzymes: The most important enzyme of this family is glutathione peroxidase which uses glutathione to detoxify ROS. Glutathione can t do so by its own; it needs the help of the enzyme. Glutathione is a tripeptide consists of: glycine, cysteine and glutamate attached to the compound by its γ-carbon instead of α-carbon. (that means glutathione has two c-terminals). When glutathione is oxidized, it forms a Disulfide bridge with another oxidized glutathione. Glutathione peroxidase requires Selenium to perform its function. But glutathione must return back to its reduced structure in order to function again, and therefore it needs the help of the enzyme glutathione reductase which uses NADPH to complete this reaction. The ONLY source of NADPH in RBCs is the pentose phosphate pathway. Therefore, any disorder in the pentose phosphate pathway will affect RBCs severely. (In other cells the pentose phosphate pathway is important but there are some other sources of NADPH like Malic enzyme(nadp+ dependent malate dehydrogenase ) and the formation of pyruvate from oxaloacetate. There are other anti-oxidant enzymes such as superoxide dismutase which reduces superoxide ions to hydrogen peroxide and oxygen. Hydrogen peroxide is removed either by glutathione peroxidase or catalase. (Catalase acts only on hydrogen peroxide and turns it into water and oxygen, while glutathione peroxidase can act on some organic peroxide as well). 9 P a g e

10 Antioxidant chemicals: Vitamin E \ Vitamin C(ascorbate) \ β-carotene (will be discussed in the next lecture). > Sources of ROS 1) Oxidases: Enzymes that Catalyze hydrogen transfer from the substrate to molecular oxygen producing hydrogen peroxide as a byproduct. 2) Oxygenases: catalyze substrate oxidation by molecular oxygen (it adds oxygen); They are either monooxegenases that transfer one oxygen atom and reducing the other to water, or dioxygenases that transfer two oxygen atoms (used in synthesis of prostaglandins and will be discussed later). >>NOTE: The difference between oxidases and dehydrogenases that both of them transfer hydrogens, but in dehydrogenases what accept these hydrogens are NAD+ and FAD and in oxidases the oxygen is the acceptor. Oxidases transfer hydrogens to molecular oxygen, but oxygenases transfer oxygen to the substrate even one oxygen or two oxygens). 3) Co Enzyme Q in the respiratory chain: produce many free radicals especially superoxide. 4) Ionizing radiation: γ or X. 5) Peroxidase: is also produces hydrogen peroxide which is responsible to produce the deadly hydroxyl free radical in the presence of iron (excess iron could be catalyst for reactions to produce free radicals like hydroxyl free radical). 6) Respiratory Burst ( during phagocytosis): O2, H2O2, OH, NO, HOCl 7) Cytochrome P450: superfamily of structurally related mixed function enzymes. (Chrome means it is colored /red because it contains a Hemegroup); we have two systems for cytochrome P450:- - Mitochondrial system: to hydroxylase steroids, Bile acids and Vitamin D. 10 P a g e

11 - Microsomal system: for detoxification of foreign compounds; Cytochrome P540 adds hydroxyl groups to make it more soluble and be eliminated. R-H + O2 + NADPH + H+ R-OH + H2O + NADP+ 11 P a g e