《生物化学biochemistry》双语课件ppt 10 糖酵解、糖异生及磷酸戊糖途径glycolysis

* 1. Glycolytic Pathway 2. Regulation of the Glycolytic Pathway 3. Gluconeogenesis 4. PPP Date lglycolysis (from the Greek glykys, meaning “sweet”） llysis, meaning “splitting” Glycolysis -is a catabolic pathway where in glucose is converted to two molecules of pyruvate with the generation of two molecules of ATP. Date Goals: - To understand the overall reaction of glycolysis - To learn the detailed 10 steps of glycolysis reaction - To know the enzyme catalyzing each step of glycolysis reaction, particularly in steps #1, 3, 6, 7,10 Date Metabolism Date Catabolism lEnergy-yielding reactions lFor non-photosynthetic organisms, two sources of fuel Diet Fats, carbonhydrates, proteins Stored compounds Fats, starch, glycogen Date Glycolysis: Embden Meyerhof pathway l1854-1864, Louis Paster, fermentation”vital force” l1897, Hans Buchner and Eduard Buchner Yeast cell-free extracts , sucrose First time that fermentation could occur outside cell The history : Date l1905 Arthur Harden and William Young Yeast cell-free extracts; Glucose; Inorganic phosphate; Heat-labile, nondialyzable component (zymase) Heat-stable, dialyzable 可透析的fraction (cozymase) Date Date l1940 , Gustav Embden, Otto Meyerhof, Carl Newberg, Jacob Parnas, Otto Warburg, Gerty Cori, and Carl Cori elucidate the complete glycolytic pathway Date Date Carl and Gerty Cori shared the Nobel Prize in Physiology or Medicine in 1947 with Bernardo Houssay of Argentina, who was cited for his studies of hormonal regulation of carbohydrate metabolism. Date The Cori laboratories in St. Louis -an international center of biochemical research 1940s-1950s six scientists got Nobel laureates: Arthur Kornberg (for DNA synthesis, 1959), Severo Ochoa (for RNA synthesis,1959), Luis Leloir (for the role of sugar nucleotides in polysaccharide synthesis, 1970) Date Earl Sutherland (for the discovery of cAMP in the regulation of carbohydrate metabolism, 1971) Christian de Duve (for subcellular fractionation, 1974) Edwin Krebs (for the discovery of phosphorylase kinase, 1991). Date Importance of Glycolysis An almost universal central energy yielding path Provides precursors for many biosynthetic paths The starting point glucose The process ends two pyruvate molecules Additional products of glycolysis include two ATPs and two NADHs Illustrates enzyme mechanisms Illustrates regulatory mechanisms Date sprints, requires a source of energy that can be rapidly ccessed. The anaerobic metabolism of glucose the process of glycolysis provides such a source of energy for short, intense bouts of exercise. Date Glycolysis lThe sequence of reactions from glucose to pyruvate acid is common to carbohydrate metabolism under both aerobic and anaerobic conditions. lGlycolysis takes place in the cytosol. Date lSome organisms (yeast) under anaerobic conditions convert pyruvate to ethanol-alcoholic fermentation lSome microorgnism (lactic acid bacteria )-convert pyruvate to lactic acid -lactic acid fermentation lIn human under aerobic conditions the end-product is pyruvic acid. Pyruvate is completely oxidized to CO2 in the TCA cycle and large amounts of ATP are subsequently produced. Under anaerobic conditions the end product is lactic acid; - - lactic acid fermentation. Date Some Points About Glucose lGlucose is very soluble source of quick and ready energy. lIt is a relatively stable and easily transported. lIn mammals, the brain uses only glucose under non-starvation conditions. lGlucose is the only source of energy in red blood cells. Date Date Date Glycolysis has 10 Steps Date Hexose stage: 2 ATP are consumed per glucose Triose stage: 4 ATP are produced per glucose Net: 2 ATP produced per glucose Each chemical reaction prepares a substrate for the next step in the process ATP is both consumed and produced in glycolysis Date Two phase First phase -预先支出阶段-ATP energy is invested Second phase -收入阶段-ATP energy is generated Date Date Date Importance of Phosphorylated Intermediates 1. Plasma membrane generally lacks transporters for phosphorylated glycolytic intermediates. 2. Phosphoryl groups are essential components in the enzymatic conservation of metabolic energy. Energy released in the breakage of phosphoanhydride bonds (ATP) is partially conserved in the formation of phosphate esters (glucose 6-phosphate). High- energy phosphate compounds (1,3-bisphosphoglycerate, phosphoenolpyruvate) donate phosphoryl groups to ADP to form ATP. 3. Binding energy resulting from the binding of phosphate groups to the active sites of enzymes lowers the activation energy and increases the specificity of the enzymatic reactions. Date 1st Stage of Glycolysis Glycolysis 1st Stage Date Step 1. Hexokinase reaction G= 16.7 kJ/mol Date Transfers the g-phosphoryl of ATP to glucose C-6 oxygen to generate glucose 6-phosphate (G6P) Mechanism: attack of C-6 hydroxyl oxygen of glucose on the g-phosphorous of MgATP2- displacing MgADP-. Four kinases in glycolysis: steps 1,3,7, and 10, all of which require Mg2+ and have a similar mechanism. Date Properties of hexokinases Broad substrate specificity - hexokinases can phosphorylate glucose, mannose 甘露糖and fructose 果糖 Yeast hexokinase undergoes an induced-fit conformational change when glucose binds 40 000倍 Date Hexokinase Reaction lRecall the “induced fit” ATP and ADP always bind to enzymes as a complex with the metal ion Mg2+. Date Date Isozymes - multiple forms of hexokinase Hexokinases I, II, III are active at normal glucose concentrations (Km values 10-6 to 10-4M) Hexokinase IV (Glucokinase, Km 10-2M) in the hepatocyte is active at higher glucose levels, allows the liver to respond to large increases in blood glucose glycogen Date Step 2. Conversion of G6P to F6P G= 1.7 kJ/mol Date Converts glucose 6-phosphate (G6P) (an aldose) to fructose 6- phosphate (F6P) (a ketose) Enzyme preferentially binds the a-anomer of G6P (converts to open chain form in the active site) Enzyme is highly stereospecific for G6P and F6P Isomerase reaction is near-equilibrium in cells, i.e., DG is close to zero. Date Step 3. Phosphofructokinase-1 (PFK-1) Reaction G= 14.2 kJ/mol Date Catalyzes transfer of a phosphoryl group from ATP to the C-1 hydroxyl group of F6P to form fructose 1,6-bisphosphate (F1,6BP) PFK-1 is metabolically irreversible and a critical regulatory point for glycolysis in most cells (PFK-1 is the first committed step of glycolysis) A second phosphofructokinase (PFK-2) synthesizes fructose 2,6-bisphosphate (F2,6BP) Date Phosphofructokinase lComplex enzyme MW 360,000 lRate-limiting step in glycolysis lMajor control point: allosteric regulation High ATP inhibits High AMP, ADP stimulates Other “fuels” alter activity Fru-2,6-bisP hormonal signal Date Radioisotopic tracer studies show: One GAP molecule: C1,2,3 from Glucose C4,5,6 Second GAP: C1,2,3 from Glucose C3,2,1 Step 4. Aldolase Reaction Date Aldolase cleaves the hexose F-1,6-BP into two triose phosphates: glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) Mechanism is common for cleaving C-C bonds in biological systems (and C-C bond formation in the reverse direction) Date aldolase reaction(the direction of fructose 1,6-bisphosphate cleavage) strongly positive aldolase reaction is readily reversible. Date Date Two classes of aldolases Class I aldolases in animals and plants, form the Schiff base intermediate Class II aldolases in fungi and bacteria, a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2 Date The class I aldolase reaction. Date Date Step 5. Reaction of Triose phosphate isomerase Conversion of DHAP into glyceraldehyde 3-phosphate (GAP) allows to be metabloized via glycolytic enzymes. Reaction is very fast Date 2nd Stage of Glycolysis Glycolysis 2nd Stage 变位酶 烯醇化酶 脱氢酶 Date G= +6.3 kJ/mol GAP converted to 1,3BPG Step 6. Reaction of Glyceraldehyde 3- Phosphate Dehydrogenase (GAPDH) Date Conversion of GAP to 1,3-bisphosphoglycerate (1,3BPG). Molecule of NAD+ is reduced to NADH Conservation of oxidative energy: Energy from oxidation of GAP aldehyde is conserved in acid-anhydride(酸酐) linkage of 1,3BPG. Oxidation of the aldehyde group of GAP proceeds with large negative free-energy change. Next step of glycolysis uses the high-energy phosphate of 1,3BPG to form ATP from ADP. Date Step 7. Phosphoglycerate kinase reaction G= 18.5 kJ/mol Transfer of phosphoryl group from the energy-rich 1,3BPG to ADP yields ATP and 3-phosphoglycerate (3PG) Date Phosphoglycerate Kinase 亲核攻击 Date Substrate-level phosphorylation - Steps 6 and 7 couple oxidation of an aldehyde to a carboxylic羧基 acid with the phosphorylation of ADP to ATP Date Substrate channel Date Step 8. Phosphoglycerate mutase 变位酶 reaction G= +4.4 kJ/mol Date The phosphoglycerate mutase reaction. Date Catalyzes transfer of a phosphoryl group from one part of a substrate molecule to another Reaction occurs without input of ATP energy Mechanism requires 2 phosphoryl-group transfer steps A covalent enzyme-phosphate intermediate (P-histidine) is involved Date Step 9. Enolase 烯醇酶(2-phosphoglycerate dehydratase脱水酶) reaction G= +7.5 kJ/mol Date 3-Phosphoglycerate (3PG) is dehydrated to phosphoenolpyruvate (PEP) Elimination of water from C-2 and C-3 yields the enol- phosphate PEP PEP has a very high phosphoryl group transfer potential because it exists in its unstable enol 烯醇 form Date Step 10. Pyruvate kinase reaction Catalyzes a substrate-level phosphorylation Metabolically irreversible reaction Regulation both by allosteric modulators and by covalent modification Pyruvate kinase gene can be regulated by various hormones and nutrients PEP + ADP Pyruvate + ATP G= 31.4 kJ/mol Date Net reaction of glycolysis Two molecules of ATP are produced Two molecules of NAD+ are reduced to NADH Glucose + 2 ADP + 2 NAD+ + 2 Pi 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O Note: all in cytosol Date The Conversion of Glucose to Pyruvate (Review) The Energy released from the anaerobic conversion of glucose to pyruvate is- -47kcal mol-1. Under aerobic conditions much more chemical bond energy can be extracted from pyruvate. Date Under anaerobic conditions pyruvate is converted to lactate. In exercising muscle The redox balance is maintained The NAD+ that is consumed in the glyceraldehyde 3-phosphate reaction is produced in the lactate DH reaction. The activities of glyceraldehyde 3- phosphate DH and Lactate DH are linked metabolically. Date The NAD+ that is consumed in the glyceraldehyde 3- phosphate reaction is produced in the lactate DH reaction. Thus, redox balance is maintained. The NADH that is produced in the glyceraldehyde 3- phosphate reaction is consumed in the lactate DH reaction. Thus, redox balance is maintained. Remember! Glucose + 2 Pi +2 ADP 2 lactate + 2 ATP + 2 H2O Date Date In anaerobic yeast, pyruvateethanol Pyruvate is decarboxylated. Acetaldehyde 乙醛is reduced. Date Industrial-scale fermentation Date Variations on a theme in alcoholic fermentation. Here also, there is no net oxidation reduction. Date What happens when we ingest what the yeast excretes. “It provokes the desire, but takes away the performance.” William Shakespeare William Shakespeare Date Date What happens when we ingest what the yeast excretes in the term of Biochemistry vEthanol is metabolized to acetaldehyde and then to acetate via alcohol DH and acetaldehyde DH. vBoth enzymes consume NAD+ and produce NADH. vThus, with the consumption of alcohol the NADH/NAD+ ratio is increased (high NADH inhibits TCA cycle) . vAlso, a significant increase in fatty acid synthesis and glycerol 3-phosphate, resulting in the accumulation triacylglycerols producing a fatty liver. Date Lactic acidosis 酸毒症 肝(实质)细胞 乙醛 双硫醒 治疗慢性酒精中毒药 Date A word on hepatic cirrhosis肝硬化 lBoth alcohol DH and acetaldehyde DH are easily saturated so a fixed quantity of alcohol is slowly metabolized; about 7g/hour. lThe accumulation of acetaldehyde 乙醛occurs with “heavy drinking”. lThe highly reactive acetaldehyde is toxic causing much of the tissue damage in chronic alcoholism急性醇中毒. It binds covalently to amino groups, nucleotides, and phospholipids to form adducts加合 物. Date Date Routes for utilizing substrates other than glucose in glycolysis 乳糖 半乳糖 麦芽糖 Date 1. Pathway of Atoms in Fermentation A “pulse-chase” experiment using 14C-labeled carbon sources is carried out on a yeast extract maintained under strictly anaerobic conditions to produce ethanol. The experiment consists of incubating a small amount of 14C-labeled substrate (the pulse)with the yeast extract just long enough for each intermediate in the fermentation pathway to become labeled. The label is then “chased” through the pathway by the addition of excess unlabeled glucose. The chase effectively prevents any further entry of labeled glucose into the pathway. problems DateDate (a) If 1-14Cglucose (glucose labeled at C-1 with 14C) is used as a substrate, what is the location of 14C in the product ethanol? Explain. (b) Where would 14C have to be located in the starting glucose to ensure that all the14C activity is liberated as14CO2 during fermentation to ethanol? Explain. DateDate 2. Glycolysis Shortcut Suppose you discovered a mutant yeast whose glycolytic pathway was shorter because of the presence of a new enzyme catalyzing the reaction: Would shortening the glycolytic pathway in this way benefit the cell? Explain. DateDate 4. Requirement for Phosphate in Ethanol Fermentation In 1906 Harden and Young, in a series of classic studies on the fermentation of glucose to ethanol and CO2 by extracts of brewers yeast, made the following observations. (1) Inorganic phosphate was essential to fermentation; when the supply of phosphate was exhausted, fermen