生物化學(xué)：Chapter 14 Glycolysis and the Pentose Phosphate Pathway

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1、To be lectured by Professor Chang Zengyi (To be lectured by Professor Chang Zengyi (昌增益教授）昌增益教授） on Nov. 20, 2012on Nov. 20, 2012Ribose 5-Ribose 5-phosphatephosphate(fat)The metabolic fates of glucose involves hundreds or thousands of chemical transformations.CO2 + H2O +ATP Go = 2840 kJ/mole+ NADPHl

2、The chemical reactions of the glycolytic pathway, fermentation, and pentose phosphate pathway.lThe elucidation of the pathways.The “l(fā)ysis” step“committed” stepBisphosphate or trisphosphate vsDiphosphate or triphosphateUsing Pi, not ATP!an acyl-phosphate (anhydride)Heavy metals (Hg2+) react with the

3、essential Cys and irreversibly inhibit the enzyme!The ending phosphate on C-2 is not the same phosphate removed from C-3. Only 5% of the potential energy of the glucose molecule is released during glycolysis.The net production of ATP per is 4-2=2.Structure of the 10 enzymesHexokinasePhosphoglucose i

4、somerasePhosphofructokinase-1AldolaseTriose phosphate isomeraseGlyceraldehyde 3-PdehydrogenasePhosphoglycerate kinasePhosphoglycerate mutaseEndolasePyruvate kinaseHexokinase Hexokinase exhibits exhibits induced induced fitfit property: The binding property: The binding of of glucoseglucose in the ac

5、tive site in the active sitecauses a major conformationalcauses a major conformationalchange.change.Inactive conformationInactive conformationActive conformationActive conformationGlucoseGlucoseSubstrate-induced cleftclosing is a general featureof all kinases!lThe realization that all the intermedia

6、tes are phosphorylated in glycolysis came slowly and serendipitously.lTo retain them in the cells (no plasma membrane transporters for them).lFacilitate ADP phosphorylation.lProvide additional binding energy (all as Mg2+ complexes; to lower the activation energy).lPolysaccharides such as starch and

7、glycogen are first degraded into glucoses via hydrolysis in extracellular spaces (catalyzed by a a-amylases and other enzymes), but into glucose 1-phosphate via phosphorolysis inside cells (catalyzed by phosphorylases).lOligosaccharides (e.g., sucrose, lactose, trehalose, etc.) are degraded into mon

8、osaccharides before further transformed.lHexoses other than glucose can also be catabolized via the glycolytic pathway after being converted to a phosphorylated derivative.Polysaccharides, disaccharides, and other hexoses are all catabolized via the glycolytic pathway. PhosphorolysisHydrolysis(lack

9、of which causes lactose intolerance)Lactobacillus bulgaricusAcidic protonThiamine (vitamine B1) Deficiency of thiamine in diet leadsto Beriberi (by eating polished rice)Christiaan Eijkman(1858-1930)Nobel Prize in 1929 vital amine NAPDH: an reducing agentand antioxidant.PPP: active in adipose tissue,

10、liver, and red blood cells; Needed for CO2 fixation in plants.Ribose 5-P: for nucleotidebiosynthesisPPP in rapidly dividing PPP in rapidly dividing cellscells: provides Ribose : provides Ribose 5-P and NADPH.5-P and NADPH.GlucoseGlycogenCyclic esterPPP in non-dividing cellsPPP in non-dividing cells:

11、 : The pentose is reconverted The pentose is reconverted to hexose!to hexose!TPPTPP轉(zhuǎn)羥乙醛酶轉(zhuǎn)羥乙醛酶轉(zhuǎn)二羥丙酮基酶轉(zhuǎn)二羥丙酮基酶GlycolysisEpimeraseIsomerase6-phosphogluconateThiamine pyrophosphate (TPP) intransketolase helps the C-C cleavage & formation (a Ping-Pong Mechanism).Compare this reaction with that catalyzed B

12、y the pyruvate decarboxylase.The cofactor-free transaldolase uses the side chain amino group of a Lys residue to form covalentenzyme-substrate intermediates, a mechanism highlycomparable to that of transketolase (also to that ofFructose 1,6-bisphosphate aldolase).lWhere cell-free biochemistry starts

13、 (Buchner, 1897) ending the vitalistic dogma and allowing metabolism to be studied in chemical terms.lWhere phosphorylated intermediates were first discovered (Harden and Young, 1900s).lWhere heat-labile, nondialyzable enzymes were distinguished from the heat-stable, dialyzable coenzymes (e.g., NAD+

14、).lWhere the unity of life being supported by the astonishing similarity between the yeast and muscle enzymes and coenzymes of alcohol and lactic acid production (“fermentation” and “glycolysis”)lWhere enzyme purification & characterization began.Fermentation proposed to be caused by the growth of m

15、icroorganisms (1860s). lUnder no circumstance can microscopic beings be born into the world without germs, without parents similar to themselves. Thus ended the spontaneous generation“ speculation. lAlcoholic fermentation is an act correlated with the life and organization of the yeast cells, not wi

16、th the death or putrefaction of the cells.“Pasteur The yeast extract was prepared for its antiseptic effects, sugar was added as a preservative but froth was observed! “Chance favors the prepared mind” (Louis Pasteur)This will bring him fame, even though he has no chemical talent! (Von Baeyer)+Tolue

17、ne(antiseptic)The discovery of the essential role of Pi and coferment in alcoholic fermentation (Arthur Harden, 1906) Coferment (cozymase later coenzyme): Dialyzable & thermostableVolumetric, instead of gravimetric, measurement of CO2!Pi is converted to organic Pi- boiled yeast juice+ boiled yeast j

18、uice (25 c.c.)+ boiled yeast juice (75 c.c.)Amount of CO2 producedequimolecular with the HPO4 added. +Pi- Pi+PiNobel prize 1929lDifficulties: The intermediates possess a very short lifetime and low steady-state concentrations.lUsing enzyme inhibitors: certain intermediates to be accumulated, isolate

19、d, and characterized.lAlkaline hydrolysis of hexoses: various 3- and 2-carbon compounds produced (the chemists way)lCriteria of judging intermediates: when added to the yeast juice (not living yeast!), fermentation rate (CO2 production) be the same, or higher than when the sugar is added.lDiscovery

20、of phosphate esters of hexoses: hexose diphosphate (1908, Harden and Young); Glc-6-P (1914, Harden and Robinson), Fru-6-P (1918, Neuberg); Recognition of the Intermediary metabolites of the Glycolytic PathwayDateStepAuthors1911Pyruvic AcidNeuberg1928AcetaldehydeNeuberg1933D-3-Phosphoglyceric acidEmb

21、den1933F-1,6-PP (Harden ester)Embden1934G-6-P (Robison ester), F-6-P (Neuberg ester)Meyerhof19342-Phosphoenolypyruvic acidMeyerhof1934PhosphodihydroxyacetoneMeyerhof1935D-2-Phosphoglyceric acidMeyerhof1936D-Glyceraldehyde-3-PMeyerhof1936G-1-PCori and Cori1939D-1,3-Diphosphoglyceric acidNegelein and

22、BrmelGlycolytic pathway also named as the Embden-Meyerhof pathwaylThe discovery of hexokinse (1927, Meyerhof) in muscle ended the idea that glucose is phosphorylated by Pi.A Chronology of the Identification of the Enzymes of GlycolysisDateEnzymeAuthors1909Alcohol dehydrogenase Bateilli and Stern1911

23、Pyruvate decarboxylaseNeuberg 1927HexokinaseMeyerhof1933Lactate dehydrogenaseAndersson1933Glucosephosphate isomeraseLohmann1934Pyruvate kinaseParnas1935Phosphoglycerate phosphomutaseMeyerhof 1935EnolaseMeyerhof 19366-PhosphofructokinaseOstern1936Fructose-biphosphate aldolaseMeyerhof1936Triosephophat

24、e isomeraseMeyerhof1936Glycogen phosphorylaseCori and Cori1936Phosphoglucomutase, glucose- phosphomutaseCori and Cori1939Triosephophate dehydrogenase, Glyceraldehyde-phosphate Dehydrogenase (NADP+)Warburg 1942Phosphoglycerate kinaseBcherGustav Embdens contributionlThe Harden-Young ester (Fru-2,6-dip

25、hosphate) increased the production of lactic acid in the juice of muscle tissue (lactic acid fermentation).lDiscovered phosphoglycerate as a fermentation intermediate.lIntuitively predicted the presence of phosphoglyceraldehyde and dihydroxyacetone, as splitting products of hexose diphosphate. lHe s

26、till could not understand the meaning of phosphorylation of the intermediates.lThe first to propose a full reaction sequence for the glycolytic pathway.lA romantic scientist (Fritz Lipmanns comment)C6H12O6 2 CH3CHOHCOOH Otto Fritz Meyerhofs contributionlThe coferments discovered by Harden and Young

27、is also required for muscle glycolysis (the first evidence on the unit of life).lIt is glycogen that is converted into lactic acid in the absence of oxygen and the lactic acid can be reconverted to glycogen in muscle (energy transformation in living cells is cyclic). lTried to understand the energy

28、transduction in muscles using the thermodynamic concepts (heat, mechanical work, free energy, phosphocreatine and other phosphorylated compounds, ATP).lATP is a cofactor participating the glycolytic pathway and will be produced as a by-product well.lRealized the importance of phosphorylation of the

29、intermediates.lPurified about one third of the enzymes.lA classic scientist (Fritz Lipmanns comment).1884-1951Nobel prize 1922Otto WarburgBernard L. Horecker lWarburgs discovery (1940s) : Glc-6-P can be oxidized to 6-phosphogluconate, a new coenzyme, NADP+, is used for this and further oxidation, an

30、d CO2 is produced (alternate pathway for carbohydrate oxidation?).lThe first product of the oxidation of 6-phosphogluconate was found to be ribulose-5-P, which can be further converted to Ribose-5-P (separated using ion exchange chromatography; using yeast and mammal preparations, Horecker, 1952).Ho

31、recker BL, (2002) “The pentose phosphate pathway”, J. Biol. Chem., 277(50):47965-71. WarburgHoreckerlThe ribose-5-P was found to be converted to hexosephosphate (i.e., being a cyclic mechanism).lRibulose diphosphate and sedoheptulose monophosphate have been identified as intermediates of CO2 fixatio

32、n in photosynthesis (Calvin).lSedoheptulose monophosphate was detected in this alternate pathway (paper chromatography).lThe catalyzing enzyme was named as “transketolase” (transferring a 2-carbon fragment from the ketopentose, ribulose-5-P; using thiamine pyrophosphate as coenzyme carrier). l lConf

33、iguration problem at carbon 3: discovery of the epimerase and xylulose-5-P.lWhat is the fate of sedoheptulose phosphate? Discovery of the “transaldolase” (transfer of an 3-carbon aldol linkage from sedoheptulose phosphate to a triose phosphate to make fructose-6-P).lFate of the remaining 4-carbons:

34、converted to Fru-6-P in a reaction catalyzed by transketolase. lThe study of glucose degradation has a rich history in The study of glucose degradation has a rich history in biochemistry (especially for enzymology).biochemistry (especially for enzymology).lGlucose is first converted into two three-c

35、arbon Glucose is first converted into two three-carbon pyruvates via the ten-step glycolysis pathway without pyruvates via the ten-step glycolysis pathway without directly consuming Odirectly consuming O2 2 and with a net production of and with a net production of two ATP molecules by substrate-leve

36、l phosphorylation.two ATP molecules by substrate-level phosphorylation.lLimited amount of energy can be released by oxidizing Limited amount of energy can be released by oxidizing glucose under anaerobic conditions by fermentation.glucose under anaerobic conditions by fermentation.lGlucose 6-phosphate can also be oxidized to form Glucose 6-phosphate can also be oxidized to form ribose 5-phosphate and NADPH via the pentose ribose 5-phosphate and NADPH via the pentose phosphate pathway.phosphate pathway.