Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway Part 1: Glycolysis This part is all Glycolysis, you need to KNOW the pathway FORWARDS and BACKWARDS. This is no joke!

Key topics: Things to Know Glycolysis Key topics: Things to Know Chemistry of each glycolytic reaction: Forwards and Backwards. The general thermodynamics of each reaction. Other sugars entry to glycolysis. What to do with Pyruvate?

Central Importance of Glucose Glucose is an excellent fuel Yields good amount of energy upon oxidation Can be efficiently stored in the polymeric form Many organisms and tissues can meet their energy needs on glucose only Glucose is a versatile biochemical precursor Bacteria can use glucose to build the carbon skeletons of: All the amino acids Membrane lipids Nucleotides in DNA and RNA Cofactors needed for the metabolism

Although not the only possible fates for glucose, these four pathways are the most significant in terms of the amount of glucose that flows through them in most cells.

Discoverers of Glycolysis (EMP Pathway) Hans von Euler-Chelpin worked out the structure of several coenzymes involve in the yeast fermentation. All got Nobel Prizes. Jacob Parnas 1884-1948

Showed that the sugar intermediates were Phosphorylated The importance of this is amazing: all sugar intermediates become phosphorylated which prevents them from diffusing out of the cell. Think about the chemistry here and ability to cross a membrane.

Glycolysis Overall 1 Glucose  2 Pyruvates + 2ATP + 2 NADH 10 Reactions: Know them Backwards and Forwards!!!! Where is this going on in a cell? For each molecule of glucose that passes through the preparatory phase (a), two molecules of glyceraldehyde 3-phosphate are formed; both pass through the payoff phase (b). Pyruvate is the end product of the second phase of glycolysis. For each glucose molecule, two ATP are consumed in the preparatory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP per molecule of glucose converted to pyruvate. The numbered reaction steps are catalyzed by the enzymes listed on the right, and also correspond to the numbered headings in the text discussion. Keep in mind that each phosphoryl group, represented here as P, has two negative charges (—PO32–). Or, does it? The “Keep in mind…” sentence came from the text…it is close but not exact, why? Now, we will go through each step (enzyme) beginning with hexokinase. EOC Problems 1+2 can be worked from this Figure and Lactate Dehydrogenase Rxn (slide 35)

Another View of Glycolysis What is This?? from KEGG Pathways Linked to Glycolysis, Enzymes by EC numbers

Human enzymes in green

Hexokinase We have already seen this enzyme several times in earlier chapters. There are several isoforms of hexokinase that we will consider later, but they all catalyze the same exothermic reaction.

Phosphohexose Isomerase Like most isomerases, the ΔGo’ is very close to zero, so there is no thermodynamic barrier or push.

Phosphofructokinase-1 (PFK-1) The second kinase in the top half of glycolysis. Note that this is pfk-1…which means there is at least another form of this enzyme (we will see it in Chapter 15). Nice exothermic reaction.

Fructose-1,6-bisphosphate Aldolase Aldolase has an interesting ΔGo’. What is the deal, does this reaction not go forward? A nice enigma. Now lets keep track of the glucose carbons in the products. See that carbon 1 of glyceraldehyde-3-P came from carbon 4 of glucose…see next slide.

Keep Track of those Carbons! EOC Problem 9..checks out the carbons from triose-phoshpates! Think about the aldolase ΔG. Because triose phosphate isomerase (TIM) converts DHAP (dihydroxyacetonephosphate) to Glyceraldehyde-3-P the 1 carbon of Glyd-3—P came for either the 3 or 4 carbon of glucose.

TIM TIM is has a low ΔGo’. After this one glucose is essentially converted to two Glyald-3-P’s.

3-P-Gyld DH This is the OXIDATIVE step of Glycolysis. It forms 1,3-bisphosphoglycerate: whose acid anhydride bond has more energy than an “ATP” and can generate ATP, see next step EOC Problem 14 is about arsenic poisoning and what happens when arsenate is involved with this reaction.

3-PGA Kinase 3-phosphoglcyerate kinase is named for the reverse reaction (look at the arrows). But after forming an ATP there is energy to burn. What happens to that energy?

3-PGA Mutase This is an isomerase type of reaction producing 2-PGA.

Enolase is a Dehydrase Now a dehyrase with a low ΔGo’ which makes this readily reversible. 2-PGA  PEP + H2O.

Pyruvate Kinase The final reaction of Glycolysis and names for the reverse reaction that just about can not go (look at the arrow). PEP phosphorylates ATP and has energy to burn. This is a big thermodynamic driver (pull) of Glycolysis.

EOC Problem 10: A look at modifying Glycolysis, could it work? A Fun Homework Problem Add up all the ΔG’o ‘s and see how: Aldolase’s endergonic ΔG is over come. Total for Glycolysis…what enzymes are doing the work? But, is that all…what about REALITY? EOC Problem 10: A look at modifying Glycolysis, could it work?

The Real ΔG Look what happens when you take into account the actual concentrations of the metabolites: the beginning kinases get more powerful, which helps reduce the endothermic block of aldolase to zero or slightly exothermic. Most from there to pyruvate are near equilibrium and can go either way…but pulled by pyruvate kinase.

Showed cancer cells had high rate of glycolysis, first to crystalize most glycolytic enzymes. Devised the first reliable, quantitative O2 uptake apparatus, lovingly called a Warburg!..but replaced by oxygen electrodes. Nobel Prize, 1931 and trained 6 future Nobel Laureates!! One of which is Hans Krebs who worked out the Citric Acid Cycle.

Medical Aspect: CANCER Most tumors: Glycolysis goes 10X faster than normal cells. And is mainly fermentative (producing lactic acid). The strategy is to: 1. Use this in Detection 2. To slow glycolysis down in cancer cells.

Hypoxia Induced Transcription Factor The anaerobic metabolism of glucose in tumor cells yields far less ATP (2 per glucose) than the complete oxidation to CO2 that takes place in healthy cells under aerobic conditions (~30 ATP per glucose), so a tumor cell must consume much more glucose to produce the same amount of ATP. Glucose transporters and most of the glycolytic enzymes are overproduced in tumors. Compounds that inhibit hexokinase, glucose 6-phosphate dehydrogenase, or transketolase block ATP production by glycolysis, thus depriving the cancer cell of energy and killing it.

Use of 6-Phospho-FdG in Positron Emission Tomography, BOX 14-1 FIGURE 2 Phosphorylation of 18F-labeled 2-fluoro-2-deoxyglucose by hexokinase traps the FdG in cells (as 6-phospho-FdG), where its presence can be detected by positron emission from 18F. Use of 6-Phospho-FdG in Positron Emission Tomography, it accumulates in regions of high glycolytic activity See next slide

CT and PET Scans Patient with malignant melanoma –ingested 6-Phospho-FdG Left-CT scan –shows location of bone, soft tissue Center-PET scan showing high glucose use. Natural areas of high glucose use: brain but not bladder Detection of cancerous tissue by positron emission tomography (PET). The adult male patient had undergone surgical removal of a primary skin cancer (malignant melanoma). The image on the left, obtained by whole-body computed tomography (CT scan), shows the location of the soft tissues and bones. The central panel is a PET scan after the patient had ingested 18F-labeled 2-fluoro-2-deoxyglucose (FdG). Dark spots indicate regions of high glucose utilization. As expected, the brain and bladder are heavily labeledﾑthe brain because it uses most of the glucose consumed in the body, and the bladder because the 18Flabeled 6-phospho-FdG is excreted in the urine. When the intensity of the label in the PET scan is translated into false color (the intensity increases from green to yellow to red) and the image is superimposed on the CT scan, the fused image (right) reveals cancer in the bones of the upper spine, in the liver, and in some regions of muscle, all the result of cancer spreading from the primary malignant melanoma. Cancer has spread to liver, muscle Right –false color composite

Type 1 Diabetes and Glycolysis Normally, insulin triggers the insertion of GLUT4 transporters into the plasma membrane by the fusion of GLUT4-containing vesicles with the membrane, allowing glucose uptake from the blood. When blood levels of insulin drop, GLUT4 is resequestered in vesicles by endocytosis. In type 1 (insulin-dependent) diabetes mellitus, these normal processes are inhibited as indicated by X. The lack of insulin prevents glucose uptake via GLUT4; as a consequence, cells are deprived of glucose and blood glucose is elevated. Lacking glucose for energy supply, adipocytes break down triacylglycerols stored in fat droplets and supply the resulting fatty acids to other tissues for mitochondrial ATP production. Two byproducts of fatty acid oxidation in the liver (acetoacetate and β-hydroxybutyrate, see p. 666) accumulate and are released into the blood, providing fuel for the brain but also decreasing blood pH, causing ketoacidosis. The same sequence of events takes place in muscle, except that myocytes do not store triacylglycerols and instead take up fatty acids that are released into the blood by adipocytes.

Entry of other Sugars The other sugars are in our diets. Mannose is in green beans, tomatoes, cranberries and blue berries. Trehalose is made by several plants, fungi and some bacteria. The sources of the others you should know before taking BCH 3033.

Glycogen Phosphorylase This is the reaction in cytoplasms, not digestive tracts. This is the enzyme that is modified by phosphorylation (phosphorylase-a and –b forms.

Entry of other Sugars The other sugars are in our diets. Mannose is in green beans, tomatoes, cranberries and blue berries. Trehalose is made by several plants, fungi and some bacteria. The sources of the others you should know before taking BCH 3033.

What is the over all Glycolytic ATP production from Fructose?

Conversion of galactose to glucose 1-phosphate Conversion of galactose to glucose 1-phosphate. The conversion proceeds through a sugar-nucleotide derivative, UDP-galactose, which is formed when galactose 1-phosphate displaces glucose 1-phosphate from UDP-glucose. UDP-galactose is then converted by UDP-glucose 4-epimerase to UDP-glucose, in a reaction that involves oxidation of C-4 (pink) by NAD+, then reduction of C-4 by NADH; the result is inversion of the configuration at C-4. The UDP-glucose is recycled through another round of the same reaction. The net effect of this cycle is the conversion of galactose 1-phosphate to glucose 1-phosphate; there is no net production or consumption of UDP-galactose or UDP-glucose.

Conversion of Pyruvate to Something Else Why to this? For aerobic cells, pyruvate is oxidized to acetyl portion of acetyl-S-CoA for entry to the Citric Acid Cycle and completely oxidized to CO2 and water (CAC + respiratory electron transport). Why under anaerobic conditions does pyruvate have to be converted (fermented) to something else = lactic acid or ethanol and CO2. The reason for this is that the needs of anabolism (many pathways together) are about 8 ATP / 1 NADH. What does glycolysis produce by converting glucose to pyruvate….is it producing too much ATP or too much NADH?

Lactate DH Lactic Acid DH uses up NADH to reduce the carbonyl of pyruvate to an alcohol in a nicely exothermic reaction. This is required to lower the NADH and provide NAD+ which is required to run Glycolysis (see next slide)

Why Lactate DH? Consider the Outputs of Catabolism and Needs of Anabolism !

Alligators do not have a great Cori Cycle Hey, what’s the Cori Cycle? This is what people do after a strenuous workout: muscles will produce lactic acid due to contractions lowering blood flow and the need to provide ATP for muscle contraction. Lactic acid makes its way to the liver which does gluconeogenesis (Part 2 of this Chapter) to convert lactic acid to glucose (this is an endothermic process). Alligators have a poor Cori Cycle and can not run long distances and when they to run, it takes them a long time to recover. Just remember this little bit of Biochemistry if a gator happens to be chasing you.

EOC Problem 6: follow the carbons from glucose into ethanol. Alcohol Fermentation Yeast when the ferment do not produce lactic acid, but rather ethanol and CO2. And look, it does the same thing, uses up an NADH ! The decarboxylase step uses TPP (next slide, and in the Pentose Phosphate Pathway, and Chapter 16) and is an oxidation. EOC Problem 4: Calculating the Keq of Alcohol Dehydrogenase (use Table 13-7) EOC Problem 6: follow the carbons from glucose into ethanol.

Entry of Glycerol into Glycolysis Only three enzymes: glycerol kinase and glycerol-3-P DH are new. Thus, to 3-P-glyceraldehyde the net yield is 1 NADH and -1 ATP. Then one 3-P-glyceraldehyde goes through the last part of glycolysis to pyruvate, see next slide.

Energetics of Glycerol as An Energy Source Glycerol kinase - ATP Glycerol-3-P DH + NADH 3-P-Gyld DH + NADH 3-PGA Kinase + ATP Pyr Kinase + ATP Total = 1 ATP + 2 NADH Glycerol as a 3 carbon sugar can not be fermented. It makes only one pyruvate, but needs to recycle 2. Therefore it can only be metabolized if there are other ways of using the “extra” NADHs…which means respiratory electron transport. Glycerol is a good aerobic carbon and energy source…respiration uses up the extra NADH. Can GLYCEROL be FERMENTED? Explain

Commercial Ethanol Fermentation Industrial-scale fermentations to produce biofuel and other products are typically carried out in tanks that hold thousands of liters of medium. EOC Problem 7…all about the heat released from fermentation…what’s going on?

Things to Know and Do Before Class Each glycolytic reaction: substrates/products, enzyme name, ΔGs. Keep track of the carbon numbers from glucose to pyruvate. Overall glycolytic ΔG. Getting other sugars and glycerol into glycolysis. EOC Problems: 1, 2, 4-7, 9, 10, 14.