Glycolysis Pathway - Biochemistry - Handout, Lecture notes of Biochemistry

Download Glycolysis Pathway - Biochemistry - Handout and more Lecture notes Biochemistry in PDF only on Docsity! Chapter 14 Glycolysis Glucose ↓glycolysis anaerobic respiration 2 Pyruvate → → → 2 Lactate (sent to liver to be converted back to glucose) Requires mitochondria and O2 ↓pyruvate dehydrogenase acetyl-CoA ↓ TCA Cycle Glycolysis is the metabolic process of converting 1 molecule of glucose to 2 molecules of pyruvate through a series of 10 enzyme catalyzed reactions. All the steps of glycolysis occur in the cytosol of a cell and do not require O2. Note: converting pyruvate to acetyl-CoA and then acetyl-CoA entering the TCA cycle indirectly requires O2. Overall Reaction: Glucose + 2ADP + 2NAD+ + 2Pi → 2 pyruvate + 2ATP + 2NADH + 2H+ + 2H2O Glucose Facts: A) Main fuel source of brain. The brain uses about 120 g of glucose per day. Only fuel source for red blood cells, cornea, lens (because these cells have no mitochondria). Kidney, medulla, testes, leukocytes, white muscle fibers use a lot of glucose because these cells have very few mitochondria. B) Starch is the storage form of glucose in plants, and glycogen is the storage form of glucose in animals. The liver and the muscles are the main storage sites for glycogen. We only store enough glycogen to survive on for about 1 day. C) The body maintains a constant level of glucose in the blood. 1) Ingest Glucose 2) Glucose passes through cells of intestinal tract and enters portal blood → bloodstream 3) Pancreas monitors the level of glucose in the bloodstream and produces hormone regulators of blood glucose levels ↓ levels too low, produces glucagons which signals the liver to put more glucose into the bloodstream ↑levels too high, produces insulin which signals cells to increase uptake of glucose and signals liver to take in glucose and store it as glycogen Glycolysis Pathway: See figure 14-1 for the glycolysis pathway. Glycolysis occurs in three stages: Priming stage- 1-3 consisting of a phosphorylation (kinase), an isomerization, and a second phosphorylation (kinase). The strategy here is to trap the glucose inside the cell and to form a compound that can be readily cleaved into symmetrical, phosphorylated three-carbon units. Splitting stage- 4-5 The strategy here is to cleave fructose into two three-carbon units that are readily interchangeable. Oxidoreduction-phosphorylate stage-6-10 consisting of a redox reaction (dehydrogenase), two dephosphorylation reactions, an isomerase (mutase), and a lyase (enolase). In this stage, ATP is harvested when the three-carbon fragments are oxidized to pyruvate. Gibbs Free Energy Changes for the Glycolytic Enzymes Rxn# Enzyme ΔG (kJ/mol) 1 Hexokinase -33.5 2 Phosphoglucoisomerase -2.5 3 Phosphofructokinase -22.2 4 Aldolase -1.3 5 Triose phosphate isomerase +2.5 6 Glyceraldehyde-3-phosphate dehydrogenase -3.4 7 Phosphoglycerate kinase +2.6 8 Phosphoglycerate mutase +1.6 9 Enolase -6.6 10 Pyruvate kinase -33.4 Step 1: 1. phosphoryl transfer (phosphate group attached to the sixth carbon) ATP-Mg2+ ––> ADP glucose —————————> glucose 6-phosphate hexokinase This reaction is coupled to the hydrolysis of ATP which results in an energetically favorable reaction (large, negative ΔG= irreversible rxn). This reaction is not unique to glycolysis. In fact, all glucose is phosphorylated once it enters the cell, no matter what its ultimate fate is (glycolysis, glycogenesis, pentose phosphate pathway). →There are two enzymes which can carry out this reaction: Hexokinase: all cells, KM = 0.1 mM, product inhibited, can phosphorylate all hexose sugars (glucose, mannose, fructose) Glucokinase: liver only, KM = 10mM, not product inhibited, specific for only D-glucose, amt. of enzyme is controlled by insulin → Cells often have transport systems built into their membranes for uptake of fuel molecules such as glucose. Phosphorylated glucose is no longer recognized by the glucose transport system and is therefore trapped in the cell. There is no transport system for phosphorylated glucose. The glucose transporter is a passive transporter. Converting glucose to G6P depletes the level of glucose inside the cell, thus keeping glucose flowing inside the cell down its concentration gradient. → Binding. Substrates must be recognized by the active site of the enzyme which operates on them. There are two factors which affect this - shape and charge distribution. The strength of the interaction can depend much on the charges. Monosaccharide have polar bonds, allowing distinctive hydrogen bonds to form. This allows an enzyme to be highly specific. However, to provide a strong interaction Step 7: formation of ATP 1,3-bisphosphoglycerate + ADP ↔ 3-phosphoglycerate + ATP Phosphoglycerate kinase Note- the enzyme is named for the reverse reaction. 1,3-bisphosphoglycerate is an energy-rich molecule with a greater phosphoryl-transfer potential than that of ATP. This formation of ATP is referred to as substrate-level phosphorylation and does not require any O2. In essence, the energy released during the redox reaction in step 6 is used to transfer an inorganic phosphate molecule onto ADP. Step 8: phosphoryl shift Phosphoglycerate mutase 3-phosphoglycerate ↔ 2-phosphoglycerate #-phosphoglycerate is not an energy-rich molecule that would be capable of transferring its phosphate group to ADP. Therefore, the molecule must undergo two rearrangements in order to form an energy-rich molecule. Step 8 is the first of these rearrangements. Here, the phosphate is simply moved from carbon 3 to carbon 2. This step is reversible, and the enzyme is used again in gluconeogenesis. Step 9: dehydration Enolase 2-phosphoglycerate ↔ phosphoenolpyruvate + H20 PEP In this step, the dehydration of 2-phosphoglycerate introduces a double bond creating an enol. An enol phosphate has a high phosphoryl-transfer potential, because the phosphate group traps the molecule in its unstable enol form creating an energy-rich molecule. When the phosphate group is donated to ADP, the enol is converted to the more stable ketone (pyruvate). Step 10: formation of ATP Pyruvate kinase PEP + ADP + H+ —————————> pyruvate + ATP In this step, the phosphate group is donated from PEP to ADP to form ATP. This step is irreversible. The enzyme is named for the reverse reaction, even though the reaction is irreversible (????). Lactic Acid Fermentation Reaction occurs in cells without mitochondria (RBC) or in cells when O2 is limited (muscle cells during exercise). The entire purpose of this reaction is to convert the NADH produced in step 6 of glycolysis back to NAD+ so that glycolysis can continue. This is simply a redox reaction in which pyruvate is reduced to lactate by the enzyme lactate dehydrogenase. Lactate can then enter the bloodstream and travel to the liver where it is converted back to glucose through the gluconeogenesis pathway. Conversion of Pyruvate to Acetyl-CoA Much more energy can be obtained from glucose if it is oxidized completely to CO2 and H2O through the TCA cycle and electron transport system (requires O2 and mitochondria). The entry to the TCA cycle is through acetyl-CoA. Pyruvate is converted into acetyl-CoA by the enzyme pyruvate dehydrogenase (multi-enzyme complex) which is located inside the mitochondria. ATP Derived from Glucose 1. No O2 available or no mitochondria: Glucose + 2Pi + 2ADP → 2 lactate + 2ATP + 2 H2O Glucose must be converted to lactate, 2 ATP produced. 2. Complete oxidation of Glucose to CO2 and H20: Conversion Products ATP formed Glucose → 2 Pyruvate 2 ATP 2ATP 2 NADH 4 ATP (α-GP shuttle) or 6 ATP(M-A shuttle) 2 Pyruvate → 2 acetyl-CoA 2 NADH 6 ATP 2 acetyl-CoA into TCA cycle 2 GTP 2 ATP 6 NADH 18 ATP 2 FADH2 4 ATP 36 or 38 ATP Regulation of Glycolysis Glycolysis is a tightly regulated pathway. Only the irreversible enzymes (hexokinase, phosphofructokinase, pyruvate kinase) can be regulated. Phosphofructokinase is by far the most regulated enzyme. Regulation can occur in several ways: 1. allosteric modulators 2. hormone control (through phosphorylation/dephosphorylation) 3. de novo synthesis Inhibitor and Toxins 1. Arsenate – affects GAP dehydrogenase Arsenate looks like Pi and is added to GAP by GAP dehydrogenase instead of Pi. The arsenate group is removed by phosphoglycerate kinase in step 7, but no ATP is generated in this step. Therefore, get 0 net ATP from glycolysis. Glycolysis is not halted, but no ATP is produced. This is detrimental to RBC. 2. Iodoacetate (iodoacetamide) – inhibits GAP dehydrogenase Inhibits GAP dehydrogenase by irreversibly binding to the Cys residue in the active site rendering the enzyme inactive. 3. Fluoride ion – inhibits enolase Fluoride ion complexes with the Mg+2 ion at the active site of the enzyme rendering the enzyme inactive. Pentose Phosphate Pathway (PPP) See Figure 14.29 in book for metabolic pathway. The primary functions of this pathway are: 1. To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells (ex: fatty acid synthesis, steroid synthesis). 2. To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids. 3. Although not a significant function of the PPP, it can operate to metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates (GAP, fructose 6-phosphate) → about 30% of the oxidation of glucose in the liver occurs via the PPP → The enzymes of the PPP are located in the cytoplasm. → The oxidative steps (1-3) generate the NADPH: For every molecule of glucose that enters the PPP pathway, 2 molecules of NADPH are regenerated from NADP+. → The nonoxidative steps of the PPP pathway are designed to generate R5P. If R5P is not needed in the cell, this 5 carbon sugar can be converted into a 6-carbon sugar (fructose-6-phosphate) and a 3-carbon sugar (GAP) which can be utilized in the glycolysis or gluconeogenesis pathways. (Note: it takes 3 molecules of R5P to make 2 molecules of F6P and one molecule of GAP) Transketolase and transaldolase are enzymes involved in the rearrangement of carbon skeletons. →Transketolase is an enzyme which transfers C2 units and requires thyamine pyrophosphate (TPP) as a cofactor. →Transaldolase is an enzyme which transfers C3 units. The reaction involves a Schiff base formation between the substrate and an essential lysine residue in the active site of the enzyme. →These two enzymes can be reversed in times when R5P need is high in the cell. PPP Regulation: See figure 14.34 The glucose-6-phosphate dehydrogenase enzyme is the main site of regulation. This enzyme is regulated by the NADP+ concentration in the cell.