1、2018/9/28,10 Glycolysis(I),2018/9/28,Glycolytic Pathway Regulation of the Glycolytic Pathway Gluconeogenesis PPP,2018/9/28,glycolysis (from the Greek glykys, meaning “sweet”） lysis, meaning “splitting”,Glycolysis -is a catabolic pathway where in glucose is converted to two molecules of pyruvate with
2、 the generation of two molecules of ATP.,2018/9/28,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, 7,10,2018/9/28,Metabolism,2018/9/28,Ca
3、tabolism,Energy-yielding reactions For non-photosynthetic organisms, two sources of fuel Diet Fats, carbonhydrates, proteins Stored compounds Fats, starch, glycogen,2018/9/28,Glycolysis: Embden Meyerhof pathway,1854-1864, Louis Paster, fermentation”vital force” 1897, Hans Buchner and Eduard Buchner
4、Yeast cell-free extracts , sucrose First time that fermentation could occur outside cell,The history :,2018/9/28,1905 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),2
5、018/9/28,2018/9/28,1940 , Gustav Embden, Otto Meyerhof, Carl Newberg, Jacob Parnas, Otto Warburg, Gerty Cori, and Carl Cori elucidate the complete glycolytic pathway,2018/9/28,2018/9/28,Carl and Gerty Cori shared the Nobel Prize in Physiology or Medicine in 1947 with Bernardo Houssay of Argentina, w
6、ho was cited for his studies of hormonal regulation of carbohydrate metabolism.,2018/9/28,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,195
7、9), Luis Leloir (for the role of sugar nucleotides in polysaccharide synthesis, 1970),2018/9/28,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 kinas
8、e, 1991).,2018/9/28,Importance of Glycolysis,An almost universal central energy yielding path Provides precursors for many biosynthetic paths The starting point glucoseThe process ends two pyruvate molecules Additional products of glycolysis include two ATPs and two NADHs Illustrates enzyme mechanis
9、ms Illustrates regulatory mechanisms,2018/9/28,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.,2018/9/28,Glycolysis,The sequence of reactions from gl
10、ucose to pyruvate acid is common to carbohydrate metabolism under both aerobic and anaerobic conditions. Glycolysis takes place in the cytosol.,2018/9/28,Some organisms (yeast) under anaerobic conditions convert pyruvate to ethanol-alcoholic fermentation Some microorgnism (lactic acid bacteria )-con
11、vert pyruvate to lactic acid -lactic acid fermentation,In humanunder 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 aci
12、d fermentation.,2018/9/28,Some Points About Glucose,Glucose is very soluble source of quick and ready energy. It is a relatively stable and easily transported. In mammals, the brain uses only glucose under non-starvation conditions. Glucose is the only source of energy in red blood cells.,2018/9/28,
13、2018/9/28,2018/9/28,Glycolysis has 10 Steps,2018/9/28,Hexose stage: 2 ATP are consumed per glucose Triose stage: 4 ATP are produced per glucoseNet: 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,
14、2018/9/28,Two phase,First phase -预先支出阶段-ATP energy is invested Second phase -收入阶段-ATP energy is generated,2018/9/28,2018/9/28,2018/9/28,Importance of Phosphorylated Intermediates,1. Plasma membrane generally lacks transporters for phosphorylated glycolytic intermediates. 2. Phosphoryl groups are ess
15、ential 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
16、 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.,2018/9/28,1st Stage of Glycolysis,Glycolysis 1st Stage,2018/9/28,Step 1. Hexok
17、inase reaction,G= 16.7 kJ/mol,2018/9/28,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 r
18、equire Mg2+ and have a similar mechanism.,2018/9/28,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,2018/9/28,Hexokinase Reaction,Recal
19、l the “induced fit”,ATP and ADP always bind to enzymes as a complex with the metal ion Mg2+.,2018/9/28,2018/9/28,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 hepatocyt
20、e is active at higher glucose levels, allows the liver to respond to large increases in blood glucose,glycogen,2018/9/28,Step 2. Conversion of G6P to F6P,G= 1.7 kJ/mol,2018/9/28,Converts glucose 6-phosphate (G6P) (an aldose) to fructose 6-phosphate (F6P) (a ketose) Enzyme preferentially binds the a-
21、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.,2018/9/28,Step 3. Phosphofructokinase-1 (PFK-1) Reaction,G= 14.2 kJ/mol,2018/9/28,Catalyzes transfer of a phospho
22、ryl 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
23、2,6-bisphosphate (F2,6BP),2018/9/28,Phosphofructokinase,Complex enzyme MW 360,000 Rate-limiting step in glycolysis Major control point: allosteric regulation High ATP inhibits High AMP, ADP stimulates Other “fuels” alter activity Fru-2,6-bisP hormonal signal,2018/9/28,Radioisotopic tracer studies sh
24、ow: 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,2018/9/28,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 bon
25、ds in biological systems (and C-C bond formation in the reverse direction),2018/9/28,aldolase reaction(the direction of fructose 1,6-bisphosphate cleavage) strongly positive aldolase reaction is readily reversible.,2018/9/28,2018/9/28,Two classes of aldolases,Class I aldolases in animals and plants,
26、 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-2,2018/9/28,The class I aldolase reaction.,2018/9/28,2018/9/28,Step 5. Reaction of Triose phosphate isomerase,Conversion of DHAP into glyceraldehyde
27、 3-phosphate (GAP) allows to be metabloized via glycolytic enzymes. Reaction is very fast,2018/9/28,2nd Stage of Glycolysis,Glycolysis 2nd Stage,变位酶,烯醇化酶,脱氢酶,2018/9/28,G= +6.3 kJ/mol,GAP converted to 1,3BPG,Step 6. Reaction of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH),2018/9/28,Conversion of
28、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
29、 of glycolysis uses the high-energy phosphate of 1,3BPG to form ATP from ADP.,2018/9/28,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),2018/9/28,Phosphoglycerate Kinase,亲核攻击,2018/9/28,Sub
30、strate-level phosphorylation - Steps 6 and 7 couple oxidation of an aldehyde to a carboxylic羧基 acid with the phosphorylation of ADP to ATP,2018/9/28,Substrate channel,2018/9/28,Step 8. Phosphoglycerate mutase 变位酶reaction,G= +4.4 kJ/mol,2018/9/28,The phosphoglycerate mutase reaction.,2018/9/28,Cataly
31、zes 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,2018/9/28,Step 9. Enolase 烯醇酶(2-phosphoglycerate deh
32、ydratase脱水酶) reaction,G= +7.5 kJ/mol,2018/9/28,3-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,2018/9/28,Step
33、 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,G= 31.4 kJ/mol,2018/9/28,Net reaction of glycoly
34、sis,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,2018/9/28,The Conversion of Glucose to Pyruvate (Review),The Energy released from the anaerobic conversion of glucose to pyr
35、uvate is- -47kcal mol-1. Under aerobic conditions much more chemical bond energy can be extracted from pyruvate.,2018/9/28,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 rea
36、ction is produced in the lactate DH reaction.The activities of glyceraldehyde 3-phosphate DH and Lactate DH are linked metabolically.,2018/9/28,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 tha
37、t 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,2018/9/28,2018/9/28,In anaerobic yeast, pyruvateethanol,Pyruvate is decarboxylated.,Acetaldehyde 乙醛is reduced.
38、,2018/9/28,Industrial-scale fermentation,2018/9/28,Variations on a theme in alcoholic fermentation.,Here also, there is no net oxidation reduction.,2018/9/28,What happens when we ingest what the yeast excretes.,“It provokes the desire, but takes away the performance.”,William Shakespeare,William Sha
39、kespeare,2018/9/28,2018/9/28,What happens when we ingest what the yeast excretes in the term of Biochemistry,Ethanol is metabolized to acetaldehyde and then to acetate via alcohol DH and acetaldehyde DH. Both enzymes consume NAD+ and produce NADH. Thus, with the consumption of alcohol the NADH/NAD+
40、ratio is increased (high NADH inhibits TCA cycle) . Also, a significant increase in fatty acid synthesis and glycerol 3-phosphate, resulting in the accumulation triacylglycerols producing a fatty liver.,2018/9/28,Lactic acidosis 酸毒症,肝(实质)细胞,乙醛,双硫醒 治疗慢性酒精中毒药,2018/9/28,A word on hepatic cirrhosis肝硬化,B
41、oth alcohol DH and acetaldehyde DH are easily saturated so a fixed quantity of alcohol is slowly metabolized; about 7g/hour. The accumulation of acetaldehyde 乙醛occurs with “heavy drinking”. The highly reactive acetaldehyde is toxic causing much of the tissue damage in chronic alcoholism急性醇中毒. It bin
42、ds covalently to amino groups, nucleotides, and phospholipids to form adducts加合物.,2018/9/28,2018/9/28,Routes for utilizing substrates other than glucose in glycolysis,乳糖,半乳糖,麦芽糖,2018/9/28,1. Pathway of Atoms in Fermentation A “pulse-chase” experiment using 14C-labeled carbon sources is carried out o
43、n 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
44、 “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,2018/9/28,(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
45、 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.,2018/9/28,2. Glycolysis Shortcut Suppose you discovered a mutant yeast whose glycolytic pathway was shorter because of the
46、 presence of a new enzyme catalyzing the reaction:,Would shortening the glycolytic pathway in this way benefit the cell? Explain.,2018/9/28,4. Requirement for Phosphate in Ethanol FermentationIn 1906 Harden and Young, in a series of classic studies on the fermentation of glucose to ethanol and CO2 b
47、y extracts of brewers yeast, made the following observations. Inorganic phosphate was essential to fermentation; when the supply of phosphate was exhausted, fermentation ceased before all the glucose was used. (2) During fermentation under these conditions, ethanol, CO2, and a hexose bisphosphate ac
48、cumulated.,2018/9/28,(3) When arsenate砷酸盐 was substituted for phosphate, no hexose bisphosphate accumulated, but the fermentation proceeded until all the glucose was converted to ethanol and CO2.(a) Why did fermentation cease when the supply of phosphate was exhausted?(b) Why did ethanol and CO2 acc
49、umulate? Was the conversion of pyruvate to ethanol and CO2 essential? Why? Identify the hexose bisphosphate that accumulated. Why did it accumulate?(c) Why did the substitution of arsenate for phosphate prevent the accumulation of the hexose bisphosphate yet allow fermentation to ethanol and CO2 to
50、go to completion?,2018/9/28,Keys: a)Alcohol fermentation requires 2 mol of Pi per mole of glucose. b)Ethanol is the reduced product formed during reoxidation of NADH to NAD+, and CO2 is the byproduct of the convertion of pyruvate to ehanol. Yes, the conversion of pyruvate to ethanol and CO2 is essen
51、tial; pyruvate must be converted to ehanol, to produce a continuous supply of NAD+ for the oxidation of glyceraldehyde 3-phosphate. Fructose 1,6-bisphosphate accumulates; It is formed as an intermediate in glycolysis. c)Arsenate replaces Pi in the glyceraldehyde 3-phosphate dehydrogenase reaction to yield an acyl arsenate, which spontaneously hydrolyzes. This prevents formation of ATP, but 3-phosphoglycerate continues through the pathway.,
