Note

1. Note • My class handouts are not intended to replace your attendance to class and/or reading something like a textbook. • Although officially lectures are mandatory I do not mind if you do not come to my lectures if you feel that this is more profitable for you. You are still welcome to come to my office and ask questions. However, do not expect me to repeat the lectures for you. • If you do not understand something (or everything) in my lectures and do not seek help it is your fault. • I will appreciate if you bring to my attention any errors (not all, please) you may find in my handouts and lectures. Suggestions for improvement are always welcomed. The best way to communicate with me is at: ferchmin@gmail.com • It is my intention to post all the handouts at http://www.ferchmin.org/ • It is assumed that you are familiar with carbohydrate structure and a lecture about that was eliminated. I placed in http://www.ferchmin.org/ an old handout about that for your reference.

2. METABOLISM This lecture is the first of a series of lectures in which the metabolism of sugars will be addressed starting with glycolysis until its catabolism to CO2 and H2O. One special activity, which I like to call brain storming, will take place the 25th and for the 2nd group the 27th of September in the afternoon to complement the lectures. The construction of a metabolic map to integrate the pathways of lipid and carbohydrate metabolism was eliminated by the Almighty LCME that considers that you have too many lectures. 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) Entry of fructose and other sugars into glycolysis. Ferchmin 2012/ metabolism /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 formulae 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 is conserved in the form of ATP.

4. 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. The TCA together with the respiratory chain produce most of the ATP.

5. What happens when somebody consumes a load of glucose? Why was the glucose high after an overnight fast? Where did the glucose go?

6. We saw that glucose was cleared from blood of a patient that took a load of glucose. What happened to the glucose? We will address the fate of glucose in several tissues that take up glucose from blood. What happens to glucose in the cells of different tissues? The biochemical transformations of the molecule of glucose in the body will be the subjects of my lectures during the following weeks.

7. 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.

9. Adipocytes and skeletal muscle are mayor players in the uptake of glucose from blood.

10. 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 Shunt Glycogen Pyruvate Lactate Pyruvate CO2 CO2 Acetyl-CoA Acetyl-CoA TCA CO2 TCA CO2 Neurons Astrocytes

11. Glycolysis is the universal metabolic pathway. It occurs, with 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 fermentation which is glycolysis. In 1897 Eduard Buchner found that cell free extracts from yeast can sustain fermentation. This opened thepossibility of in vitro study of biological processes and the beginning of biochemistry. In 1905 it was found that a heat-sensitive high-molecular-weight enzymes and a heat-insensitive low-molecular-weight cofactors are needed for fermentation to proceed. The main features of the pathway were determined by 1940. New features were discovered in the last few decades.

12. G-6-P is not committed to glycolysis GLYCOLYSIS First stage, priming. 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

13. 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. 

14. Phosphofructokinase and triosephosphate isomerase cause a % distribution of fructose-1,6-di-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. 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. (67%) (3%) (30%)

15. 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

16. 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.

18. We will consider in detail, some aspects, of several of the steps of glycolysis 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 You should understand the biological meaning of all allosteric inhibitors and activators in all pathways. Try to understand not to cram!

21. Regulation of glucokinase by fructose and the role GKRP As mentioned 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 then inhibits and pulls the GK into the nucleus. Dietary fructose (like from fructose enriched corn syrup) is converted to fructose-1-phosphate, which displaces fructose-6-P, inhibits GKRP and frees GK to leave the nucleus. By this mechanism fructose-1- phosphate could stimulate lipid synthesys.

22. Regulation of phosphofructokinase I

23. 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, is not the principal toxic effect of As. We will see later its acute lethal effect on lipoic acid.

24. 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?

25. Do not to be confuse phosphoglycerate mutase with bisphosphoglycerate mutase which 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.

27. The alternative to lactate formation is to produce alcohol

28. Metabolism of alcohol Are you aware of the poisoning with car coolant (ethylene glycol) and how to deal with it?

30. Did you know that fructose and high fructose corn syrup makes you FAT, causes gout, e.t.c.

31. Low Km, saturates at low fructose concentration 10 time faster than glucokinase, high Vmax Fructose-1-P aldolase is also called aldolase B. It has low capacity, saturates rapidly. There are isozymes A,B and C . Fructose-1-P accumulation causes hypophosphatemia, hypoglycemia and gout. Why? Glyceraldehyde-kinase The glycolytic intermediates can go to glycolysis (FAT) or glycogen

33. Entry of glycogen into glycolysis • What is the net yield of ATP in glycolysis? • When glucose comes from free glucose. • When it comes from glycogen. • When 100% of metabolites go through the 2,3-diphosphoglycerate shunt. • d) When only arsenate (not phosphate) intervenes in the glyceraldehyde dehydrogenase reaction?

35. 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 following lectures.