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Ferchmin 2016 Introduction to metabolism in general and of carbohydrates in particular It would have been advantageous if you had reviewed or studied the structure of carbohydrates before the upcoming lectures. 1 Note I will appreciate if you bring to my attention any errors you may find in my handouts and lectures (not all at once, please). Suggestions for improvement are always welcomed. The best way to communicate with me is at [email protected] I never check [email protected] My “home made” web page is www.ferchmin.org I post all my handouts at http://www.ferchmin.org/MedStudents.html I must assume that you are familiar with carbohydrate structures. You can study this topic either using the SIM or my Carbohydrate Structure pptx file. My handouts are not meant to replace books authored by a team of specialist and edited by multimillion dollar companies. The objective of my handouts is to help me to present the subject and to clarify doubts you may have. The final goal is to prepare you to understand the courses that follow in the next semesters and to pass the external exams. The scope of National Board Exams is wrapped in the mist of the American “burrocracy” (misspelling intended), so I can only use my previous experience and intuition to guess what they will ask you in the NBE. 2 METABOLISM Summary of this handout: 1) General overview of metabolism. What is life? 2) Metabolic pathways. Anabolic and catabolic pathways. 3) Fate of glucose in: a) whole organism. b) specific types of cells. 4) The glycolytic pathway. 5) Regulatory enzymes of glycolysis. 6) Closer look at selected glycolytic enzymes. 7) Alcoholic fermentation and brief description of the metabolism of ethanol. 8) Pathway of carbon in glycolysis. Use of 14C in biochemistry. 9) Metabolism of fructose and entry into glycolysis. 10) Entry of other sugars into glycolysis. 3 Metabolism is the set of chemical reactions that takes place in an organisms. It provides energy (from food or other sources), synthesizes and degrades the molecules that form the organism. Life could be defined as a system of steady state reactions that take place in an open system and is endowed with the potential capability of producing similar systems. For the sake of didactics, metabolism is divided into more or less arbitrarily defined pathways. Beware, however, that different pathways often share metabolites. For every pathway you ought to know: 1) Purpose of the pathway. (Adaptive value for the organism). 2) Molecules going in and coming out? (The starting metabolites and the final products). 3) Place where it happens (organs, types of cell, subcellular compartments). 4) Regulatory enzymes. (Metabolic conditions that stimulate or inhibit the pathway). 5) Organization of the pathway and the formulas of the compounds involved. (The map of the pathway). 6) Relationship with other pathways. (Shared metabolites, enzymes and regulations). 7) Later, you will have to visualize each pathway interacting with other pathways in normal and in pathological conditions. Anabolic reactions consume energy and nutrients to synthesize cell components like proteins. Catabolic reactions break down complex molecules and release the energy which 4 is conserved in the form of ATP. Complex molecules are in equilibrium with its precursors The precursors can be incorporated into large macromolecules or catabolized. Many metabolites are converted into acetyl-CoA which is the “fuel” of the TCA. The TCA together with the respiratory chain produce most of the ATP. 5 What happens when somebody consumes a load of glucose? Glucose tolerance test Blood Glucose mg/100 ml 300 Severe diabetes 250 200 Why was the glucose high after an overnight fast? Moderate diabetes 150 100 Where did the glucose go? Normal 50 0 1 2 3 Hours after 100 g of oral glucose 6 METABOLISM OF GLUCOSE The metabolism of glucose is different in different cells. Red blood cells, neurons, liver parenchymal cells, intestinal mucosa, kidney tubular cells, eye lens cells, cornea, testis, and leucocytes are among those that do not require insulin for glucose uptake. However, even for those cells insulin is important for other reasons. 7 8 Adipocytes and skeletal muscle are mayor players in the uptake of glucose from blood. 9 It seems that astrocytes use glucose and produce lactate which is used as energy source by neurons. There is still some disagreement about that. Glucose (insulin independent) Glucose (insulin independent) Glucose-6P Glucose-6P Pentose Phosphate Pyruvate Shunt Glycogen Lactate CO2 Acetyl-CoA TCA Neurons Pyruvate CO2 Acetyl-CoA CO2 TCA CO2 Astrocytes 10 Glycolysis is an universal metabolic pathway. It occurs, with minor variations, in nearly all organisms, both aerobic and anaerobic. The wide occurrence of glycolysis indicates that it is one of the most ancient known metabolic pathways. In 1860 Louis Pasteur discovered that microorganisms are responsible for alcoholic fermentation which is a minor variation of mammalian glycolysis. In 1897 Eduard Buchner found that cell free extracts from yeast can sustain fermentation. This opened the possibility of in vitro study of biological processes and the beginning of biochemistry. In 1905 it was found that a heat-sensitive high-molecular-weight molecules (enzymes) and a heat-insensitive low-molecular-weight (cofactors) are required for fermentation to proceed. The main aspects of the pathway were determined by 1940, however, new features are being discovered as we speak . The following slides are a detailed presentation of glycolysis. 11 GLYCOLYSIS First stage, priming. G-6-P is not committed to glycolysis Glycolysis has two stages. The first primes (prepares) glucose “wasting” 2 ATP in the process. Glycolysis is “supposed to” produce ATP. Committed step of glycolysis 12 Continuation of the priming stage of glycolysis The equilibrium constant for triose phosphate isomerase favors dihydroxyacetone phosphate, but since only glyceraldehyde-3-phosphate is capable to enter glycolysis by subsequent reactions all the carbons from glucose eventually become glyceraldehyde 3-phosphate. 13 Phosphofructokinase and triosephosphate isomerase cause a % distribution of fructose-1,6di-P (30%), dihydroxyacetone-P (67%) and glyceraldehyde-P (3%). The inactive metabolite accumulates as a “reserve” while the active one is present in lower concentration. (67%) The active metabolite glyceraldehyde-3-P is present in lower concentration. It is more manageable to control a small pool than a huge one. This “strategy” is used by other pathways. That is the active metabolite is scarce. (3%) (30%) 14 Second stage, ATP production from glucose. High energy phosphate What happens if NAD+ is exhausted? 2 moles of ATP per mole of glucose. This kinase is reversible This is the first substrate level phosphorylation "site". ΔG°'= - 4.5 Kcal/mole 15 Continuation of second stage, ATP production from glucose. Another high energy phosphate The second substrate level phosphorylation is done by pyruvate kinase Glycolysis can be totally anaerobic. In that case, to continue glycolysis NAD+ must be regenerated at the expense of pyruvate that becomes lactate. Lactate is transferred from peripheral tissues to liver Lactate accumulates in muscle during exercise and in milk during fermentation like in yoghurt production. 16 17 We will consider in detail, some aspects, of several steps of glycolysis Hexokinase is not the committed Committed and rate limiting step of glycolysis!! There is a phosphofructokinase II that is not a glycolytic enzyme. Phosphofructokinase II synthesizes fructose-2,6-P step because glucose-6-P can go to the pentose shunt, glycogen synthesis, and is a precursor of inositol and glucuronic acid. You should understand the biological meaning of all allosteric inhibitors and activators in all pathways. Try to 18 understand not to cram! Glucokinase is not inhibited by G-6-P, however it is inhibited by fructose-6P. Interestingly, the inhibition by fructose-6P is reversed by fructose-1P. We will discuss the dietary importance of this and of fructose enriched corn syrup. 19 Regulation of glucokinase by fructose and the role GKRP As indicated in the table shown before, glucokinase (GK) is not inhibited by glucose-6-P. However, it is inhibited indirectly by the next glycolytic intermediary, fructose-6P. Fructose-6P binds to the glucokinase regulatory protein (GKRP) which is both an inhibitor and a nuclear receptor for glucokinase. GKRP bound to fructose-6P sequesters and inhibits GK translocating it into the nucleus. Dietary fructose (like from fructose-enriched corn syrup) is converted to fructose-1-P. Fructose-1-P displaces fructose-6-P and allows the free active GK to leave the nucleus. By this mechanism, fructose-1-P increases the concentration of glucose-6-P in the liver and stimulates the 20 synthesis of fatty acids and of purines. Comparison of glucose regulation of hexokinase and glucokinase 21 Regulation of phosphofructokinase I 22 Mechanism of action of glyceraldehyde 3-phosphate dehydrogenase: large circle represent the enzyme, small circle, the binding site for NAD+. This is not a regulatory enzyme. If arsenate is substituted for phosphate then 3-phosphoglyceroyl-arsenate is formed. The latter molecule decomposes spontaneously and no high energy bond is conserved for ATP synthesis. This, however, is23 not the principal toxic effect of As. We will see later its acute lethal effect on lipoic acid. The phosphoglycerate shunt and 1,2-diphosphoglycerate As mentioned before this is the first substrate level phosphorylation "site". ΔG°'= - 4.5 Kcal/mole What 2,3-diphospho glycerate does to hemoglobin? What is the concentration of 2,3dPglyc in erythrocytes? What is the net yield of ATP/mole of glucose in glycolysis if 100% of glucose goes through the diphosphoglycerate shunt? 24 Do not confuse phosphoglycerate mutase with bisphosphoglycerate mutase The latter catalyzes the conversion of 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate. In the enzyme's initial state, the active site contains a phosphohistidine complex formed by phoshphorylation of a specific histidine residue. 25 26 The alternative to lactate formation is to produce alcohol 27 From: http://library.med.utah.edu/NetBiochem/tabletit.htm#carbos 28 Metabolism of alcohol Are you aware of the poisoning with car coolant (ethylene glycol) and how to deal with it? 29 Use this sketch to follow the movement of labeled carbons from one metabolic pathway to another. A question you could find later in this course might be: Starting fatty acid synthesis from glucose-3-C14. In which positions of a newly 30 synthesized fatty will the C14 be found? Low Km, saturates at low fructose concentration FRUCTOSE METABOLISM 10 time faster than glucokinase, high Vmax Hexokinase muscle low Km Fructose Fructose-6P usually from sucrose about 15-20 % of calories (candies?) If missing Essential fructosuria BENIGN Liver Fructokinase Fructose-1-P aldolase is also called aldolase B. It has low capacity, saturates rapidly. There are isozymes A,B and C . Fructose-1P Fructose-1-P ALDOLASE Fructose-1P kinase SLOW Glycolytic intermediate Fructose-1,6P Fructose-1-P accumulation causes hypophosphatemia, hypoglycemia and gout. Why? If fructose-1-P aldolase is missing there is liver failure. Fructose intolerance Aldolase isozyme CHO HCOH CH2OH CH2OH CO CH2O-P ic yt ol P c y e Gl iose eras tr o m is ATP Glyceraldehydekinase Ratio of activity on fructose-1,6diphosphate and fructose -1phosphate A (Glycolytic) 13 B (Hepatic) 0.9 C (Hepatic) 7 Fructose-1,6-DP glycolytic intermediate CHO HCOH CH2OP Glycolytic Aldolase https://cheminfo2011.wikispaces.com/Anthony+Lloyd+II+-+Final+Project The glycolytic intermediates can go to glycolysis (FAT) or glycogen 31 32 Entry of glycogen into glycolysis What is the net yield of ATP in glycolysis? a) When glucose comes from free glucose. b) When it comes from glycogen. c) When 100% of metabolites go through the 2,3diphosphoglycerate shunt. d) When only arsenate (not phosphate) intervenes in the glyceraldehyde dehydrogenase reaction? 33 Overview and future projection: Notice how Glucose relates to HMP pathway (PPP) and to glycogen. Visualize the NADH cycle. The formation of CH3—CO~CoA links glycolysis to FAT synthesis and entry to the TCA or Krebs's cycle. In the following lectures we will study the synthesis of glucose from lactate and other sources. Study glycolysis NOW to be able to understand the next lecture. 34