MCB102-3 - MCBIOZ Fall 2008 Feeder Pathways for Glycolysis...

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Unformatted text preview: MCBIOZ Fall 2008 Feeder Pathways for Glycolysis. Gluconeogenesis. The Pentose Phosphate Pathway Reading: pages 543-564. 0 utline 2 1. 2. 9°.“S339” 9. Glycogen is broken down to glucose 1-phosphate, which becomes glucose 6-phosphate. Fructose can be phosphorylated to fructose l-phosphate, which is cleaved by a special aldolase, which can be defective and cause fructose intolerance. Lactose is hydrolyzed to glucose and galactose by lactase, which may not be expressed in adults. This problem leads to lactose intolerance. Galactose requires three enzymes in order to be transformed into glucose 1-phosphate. A defect in any of these three enzymes leads to a serious condition called galactosemia. Gluconeogenesis means a new creation of glucose. It occurs in the liver and kidney. It uses the seven reactions of glycolysis that are reversible. It uses four enzymes that are not used in glycolysis to provide a favorable energy balance. The pentose phosphate pathway or pentose phosphate shunt uses the energy of glucose catabolism to make NADPH for biosynthesis. 10. This pathway has an oxidizing part and a carbon rearrangement part. 11.A mutation in the oxidative part of this pathway gives resistance to malaria and sensitivity to fava beans. 12.A mutation in the carbon rearrangement part of this pathway gives a nutritionally-triggered neurological disease called The Wernicke- Korsakoff Syndrome. Gl co en de adation is a feeder athwa for 1 col sis Glycogen is a storage form of glucose that is found as large granules in muscle and liver. It is a polymer of glucose with no real reducing end, because it begins with a glucose residue whose carbon-1 is in glycosidic linkage to a protein called glycogenin. This polyglucose molecule has on 1-94 linkages and branches with oz 196 linkages: HOLH1 Nonreducing end figs . ,0 °“ l1) CH “OCH 0 1 ‘ i 1 H005; R H Rfigucing .r-o ° 0 0“ GLYCoGENW WWQ 0 o 0 $0 H H H 2,000 ends. The structure is a loosely packed helix, into which enzymes can easily penetrate. ’ In order to investigate glycogen breakdown, Carl and Gerty Cori isolated glycogen granules and looked for their breakdown in crude cell extracts from liver and muscle. The reaction that they found was greatly stimulated by phosphate, and when they purified the enzyme they found that the reaction was absolutely dependent upon phosphate, and that the product was glucose 1-phosphate: H HofiHg H00 1 o + ' H o L b-g—o‘ ? 11L @qu H G-l 4 1 .. 0 0903 O C} 7:034:91 0 H O H a“ H Since this enzyme uses phosphate to produce glucose 1-phosphate, they called it glycogen phosphorylase. At equilibrium, they found 9432 2 [Pi ]/[glucose 1-phosphate] = 3.6 (Since glycogen granules are not very soluble, we ignore their concentration). This ratio dictates that phosphate should be the product and glucose 1-phosphate should be the substrate. However, the concentration of phosphate in cells is always about 100 times the concentration of glucose 1-phosphate, because cells have an active transport mechanism for pumping in phosphate ions, and glucose l-phophate is removed by metabolism. According to Le Chatlier’s principle, any change from equilibrium results in a shift toward equilibrium, and thus the high phosphate concentration in cells causes the reaction to go toward glucose 1- phosphate. Glucose-l-phosphate can be converted to glucose 6-phosphate by the enzyme phosphoglucomutase. " OCH; 0Q“ ’PHOSPHO’ » P H a GLuCO- (2% ‘g H MuTnSE : N 0 a 0P03 V’M o H o H a H Most of what is known about glycogen metabolism comes in the next chapter. In this chapter, the authors only make the point that glycogen feeds glucose 6-phosphate into glycolysis. Fructose intolerance In muscle, hexokinase phosphorylates fructose to fructose 6-phosphate. In liver, the isozyme of hexokinase is called glucokinase, and it does not phosphorylate fructose. Instead, fructose is phosphorylated by fructokinase, which makes fructose l-phosphate: . Hod” u no €03 ’ “echo HcoH , Lp'bifitoKW’iSE P o “o + RTP ———————~—~=> Ab "' "o _ V." 0 fl H H The fructose l-phosphate is converted to glyceraldehyde and DHAP by fructose phosphate aldolase: .' ’0 Wm H1C0P03 ’1’?“ ”L90” “o r: “(30” 1’ ‘ {:6 .. 0 ll HLC 0 H HZCO P03. H . GLycgfiflLDEHYDE "DH/l? This enzymatic reaction is the rate-limiting step for entry of fructose into glycolysis, especially in people with partially defective fructose phosphate aldolase. Babies with such a mutation start to vomit when they stop nursing (34232 3 and eat fruit, which contains a lot of fructose. Fructose 1-phosphate accumulates, and the liver runs short of phosphate. At one time, physicians tried feeding fructose intravenously instead of glucose. This led to a buildup of fructose 1-phosphate and shortage of phosphate in liver, even for normal people. Thus fructose is no longer fed intravenously. Lactose intolerance Lactose (after the Latin verb “lactare,” to suckle) is a common disaccharide found in human mothers’ milk and in cows’ milk. It contains D- galactose in glycoside linkage to D-glucose in a B linkage between carbon-1 of galactose and carbon-4 of glucose. H0 H1 “00‘" 0 0H LACTOSE Ho 0“ Om '3 ANOMER a 0H Infants can hydrolyze it to glucose and galactose using an enzyme called “lactase.” In most human populations lactase is absent in adults. However, in northern Europe, survival depended upon using cows’ milk and cheese to survive the winter, and thus a population was selected that has lactase in 97% of adults in Denmark. Danes might feed milk or ice cream to people who just flew in from Thailand, and after a few hours, the lactose would have passed through their stomachs and small intestines without hydrolysis, only to be digested by bacteria in the large intestine, that produce carbon dioxide, methane and hydrogen gases. To avoid this problem, one can buy milk that contains bacteria that produce an enzyme, B-galactosidase, which hydrolyzes lactose to glucose and fructose. WM How galactose enters glycolysis D- galactose, usuallv derived from lactose, must undergo extra enzymatic reactions to enter the glycolytic pathway: H0 O H HO O H O H OH H 1 tk OH H OH H H OH gaacomase 01303 0—— P 0—— P o Uridine H OH 0% Galactose Galacfose-l-phosphate UDP—g‘lucoose galactose—l-phosphate UDP—galaetosev 3 2 uridylyl transferase 4-epimerase N AD+ CHZOH CHgOH H O H HO O H OH H OH H HOPOJ/ 0— P—~ 0— P 0.. Uridine Glucose-1 1-phosphate (GlP) UDP-galacotose 4 phosphoglucomutase CH20P03 H O H OH H Glucose-G-phosphate (GGP) FIGURE 17-36 Metabolism of galactose. Four enzymes participate 1n the conversion of galactose to the glycolytic intermediate G6P: (1) galactokinase (2) galactose—I- phosphate uridylyltransferase (3) UDP galactose-4- -epimerase and (4) phosphoglucomutase UDP (3 e c7 N MRIDIUE DIPHOS’PHATE diode-gen, Galactose is transformed to galactose 1-phosphate by galactokinase. Galactose 1-phosphate uridylyl transferase transfers the galactose 1- phosphate onto uridine diphosphate glucose to give UDP-galactose and glucose 1-phosphate. UDP-galactose-4-epimerase transforms UDP-galactose into UDP-glucose, which can start the cycle over again. The epimerase uses NAD+ to oxidize carbon-4 and to re-reduce it to yield the opposite configuration. The net result of this series of reactions is to transform galactose into glucose 1-phosphate, which can be transformed into glucose 6- phosphate by phosphoglucomutase. Glucose 8-phosphate can then enter the glycolytic pathway. There is genetic evidence for the importance of the enzymes in this pathway. Bacteria such as Escherichia coli can use galactose as their sole source of carbon and energy, and they can produce these enzymes. When mutant bacteria are isolated that cannot grow with galactose as sole source of carbon and energy, they are found to be missing at least one of these enzymes. There is a human genetic disease, called galactosemia, which exhibits deficiencies in this pathway. Most babies start to nurse a day after birth and digest lactose and galactose. Rare babies start to cry and vomit when they begin nursing, because they are deficient in galactokinase or transferase or epimerase. These babies are said to be galactosemic, and they have mutations in both copies of the same gene. Such babies are fed a formula lacking galactose. If they do not stop nursing immediately they develop cataracts from the accumulation of galactitol in the lens of the eye; they lose IQ points, and they can become mentally retarded for reasons that are not understood. The most serious mutation is in the transferase gene on the short arm of chromosome 9 at a position called P13. Babies with such a mutation do not thrive even on a low-galactose diet. Gluconeogene sis Gluconeogenesis means the “new creation of glucose” in Greek. The supply of glycogen in the liver can only last for approximately a day, and the brain functions best with glucose as its energy supply, so the liver and kidney are equipped to produce glucose from smaller molecules, but only from three carbon compounds, such as pyruvate and lactate, and not from two carbon compounds, such as acetate. Many amino acids can be broken down to yield pyruvate, and when 14C-labeled amino acids have been fed to rats that are starved for glucose, the label has been found to appear in glucose. The pathway of gluconeogenesis uses the reactions of glycolysis that are reversible and circumvents the others, using new reactions that cleave a high-energy phosphate bond. {war 6; Text Table 14-2 Free-Energy Changes of Glycolytic Reactions in Erythrocytes Glycolytic reaction step AG” (kJ/mol) AG (kJ/mol) §§®tlhicose + ATP —-—> glucose ti-pirosplmtc + Alli" » my ....) W333; if @j: Glucose 6—phosphate 3:: fructose (ii—phosphate 13,7 0 lo 25 M {3} li‘mctosc 6—pl‘rospl‘iate + A'l‘l~"—~—-> fr'uctOsc l,{i—blfiliilloSPllHLC + Alli) whiz "é ”-333 @ Fructose LES—bisphosphete :2 dihydroxyacotone phosphate + 23.8 ~6 to O glyceraldehyde B-phosphate (5] Dihydroxyacetone phosphate : glyceraidehyde 3—phosphatc 7 0 to 4 _ @ Glyceraldehyde 8—phosphate + Pi + NAD+ ,_—*.__ 1,8—bisphosphoglycerate + NADH + H+ 6.8 ”2 to 2 @ 1,B-Bisphosphoglycerate + ADP m 3-phosphoglycerate + ATP _ 188 0 to 2 @ 3-Phosphoglycerate ct: 2—phosphoglycerate 4.4 0 t0 0-8 @ 2—Phosphoglycerate :2 phosphoenolpyruvate + H20 7.5 0 to 3.3 16.7 _ )Phosphoenolpyruvate + ADPw» pyruvate + ATP ~8l.4 '9 — Note: AG” is the standard free-energy change, as defined in Chapter 13 (pp. 491—492). AG is the free-energy change calculated from the actual concentrations of glycolytic intermediates present under physiological conditions in erythrocytes, at pH 7.The gly- colytic reactions bypassed in gluconeogenesis are shown in red. Biochemical equations are not necessarily balanced for H or charge (p. 501). Looking at the standard free energy release, the irreversible reactions appear to be (1) hexokinase, (2) phosphofructokinase, (7) phosphoglycerate kinase, and (10) pyruvate kinase. Looking at the estimated real free energy changes in cells where ATP levels are high, the phosphoglycerate kinase reaction appears to be reversible, so only three glycolytic reactions are irreversible. These values of AG in this table are based on erythrocytes, and gluconeogenesis occurs in liver and kidney, so the values given may not be precise. It appears that if one could make phosphoenolpyruvate, making glucose would be feasible. The first new reaction of gluconeogenesis is the pyruvate carboxylase reaction, which depends upon the Vitamin biotin as a cofactor. 9 75mm” in AMibE LINKAGE HN/C\NH To A Lyswz SIDE CHAIN l l Her-Till f" H l \N-—-L 5 ll /\/\/ Y “icy/C o 0 BMW 0 o 5? ~ I .. 3 H ATF+HC03..,ADP+0-E-oaéco~ o—f—ol—l "o'C-N’C\NH 'o 0' . eqrhorprGSqule “ H 0 H H R . r ”0 81017” H 5/ E) O O-ECHL‘fi‘c‘O- 6K H3C‘5“C{CO' o omioacci’q’rf pygmy}; 2'" pafié f Acetyl-CoA stimulates the pyruvate carboxylase reaction for the following reason. This reason may not be clear to you, since you may not remember the citric acid cycle from Biology 1A. When oxaloacetate is depleted, acetyl-CoA accumulates, since the citrate synthase reaction of the citric acid cycle requires oxaloacetate. Therefore, acetyl-CoA must stimulate oxaloacetate synthesis in order to make the citric acid cycle function. The pyruvate carboxylase reaction occurs in mitochondria where the citric acid cycle occurs. Pyruvate carboxylase is also the first step in gluconeogenesis. When fatty acids are breaking down to acetyl—COA, it is also likely that glucose is needed. Thus activation of pyruvate carboxylase by acetyl-COA serves both functions of pyruvate carboxylase. Biotin is required as a cofactor in the pyruvate carboxylase reaction. It is carboxylated in the first step of the reaction. Carboxylated biotin donates its carboxyl group to pyruvate in the second step of the reaction. Two different domains of the enzyme catalyze the two seeps of the reaction. Biotin has a flexible side chain that is covalently attached to a lysine side chain in amide linkage. In the active state of the enzyme (with acetyl-CoA bound), biotin can swing back and forth between the two catalytic domains. In the inactive form (without bound acetyl-CoA), the two catalytic domains are too far from biotin to be active. In the figure below, only one of the four identical subunits of the pyruvate carboxylate tetramer are shown. The structural work on pyruvate carboxylase is described in Science 817:1076 (2007). BMW CflKfioflL mom) +. CAKBoX‘IL cmm‘l ‘WWSFE‘t CAKWYL‘ 3'9““ TRANSFER ATM" gulf“ BTMN z ACTIVE ENZYME “2:12:52 (Bonn/D Acem-oA) Par 3 The next reaction of gluconeogenesis occurs in the cytosol, so oxaloacetate needs to be transported out of the mitochondria. There is no transport system for oxaloacetate in mitochondria, so oxaloacetate is reduced to malate, which is transported out, and malate is re-oxidized to oxaloacetate in the cytosol. “MM” N N ”ugh! Nfibmm OXHLo ACETArgL—zMLME MAL ATE OXALO ACETATE ’3» ,P 07>ch CH-‘C‘O . MITOCHONDRwN MmeRME CYTOSoL The next bypass reaction used to reverse glycolysis converts oxaloacetate to PEP and carbon dioxide and cleaves a high-energy phosphate bond. PE? '9 P03 (3 ,4 f? .f’ - cnkaoxykmasr o o GTP +6'C‘ C~ C—C’O l N H -———————-—-———-—') C033 GDP «k HC’C’C‘O“ Jre onlocKQ‘L“ “PEP Once PEP has been made, glycolysis can be run backwards until the PFK-l reaction, which releases quite a bit of free energy when run in the direction of glycolysis. This reaction is circumvented by a phosphatase reaction. Fructose 1,6 bisphosphate is hydrolyzed to fructose 6-ph0sphate by the enzyme fructose-bisphosphatase-1 (FBPase 1). Fructose 6-phosphate is converted to glucose 6-ph0sphate and then hydrolyzed to glucose by glucose 6-phosphatase, which is bound to the endoplasmic reticulum and Which promotes release of glucose into the bloodstream. In terms of energy balance, gluconeogenesis costs 2 ATP per glucose in the pyruvate carboxylase reaction, 2 GTP in the PEP carboxykinase reaction, 2 ATP in the phosphoglycerate kinase, and 2 NADH in the GAP dehydrogenase reaction. WC? Glycolysis Gluconeogenesis ATP Glucose. P; hexokinase giumsn (imhosphamsr ADP Glucose 6-phosphate H20 1) ATP Fructose 6-phosphate fructose phospho- 1.6-bisphnsphatase»1 fructokjnase») ADP Fructose 1,6-bisphosphate H20 Dihydroxyacetone Dihydroxyacetone phosphate phosphate \ / (2) Glyceraldehyde 3—phosphate (2) P1 (2) Pi (2) NAD‘ (2) NAD“ (2) NADH + (2) H‘ (2) NADH + H+ (2) 1,3-Bisphosphoglycerate (2) ADP (2) ADP (2) ATP (2) ATP (2) 3-Phosphoglycerate JP (2) 2-Phosphoglycerate Jr (2) GDP (2) Phosphoenolpyruvate (2) ADP PEP carboxykinase Pymvate kinase (2) GT? (2) Oxaloacetate (2) ATP (2) ADP pyruvate carboxylase (2) Pyruvate (2) ATP FIGURE 14—16 Opposing pathways of glycolysis and gluconeogene- sis in rat liver. We reacticms of glycotysis are on. the )efl side, in red; the opposing pathway“ Gf gguconeogenesis és on the right, in bfue‘ The magor sizes of regulation of giuconeagenesis Shawn here are d§scussed later in this chapter, and in detail in Chapter 15. Fégure 14—39 éilustrates an aitemative route for oxatoacetate produced in mitochondria. {301%110 The pentose phosphate pathway The pentose phosphate pathway, also known as the pentose phosphate shunt, causes oxidation of glucose and reduction of NADP+ to provide reducing power for biosynthesis of molecules such as fatty acids. The entire pathway is summarized on the next page. The activity of this pathway is high in adipocytes and liver, where fatty acids are made. Otto Warburg discovered the first enzyme of the pentose phosphate pathway, using the spectrophotometer that he had invented, to measure the reduction of NADP+ to NADPH, which absorbs at 340 nm. Warburg used red blood cells, where o o o 51 If H ,H n H, H p; C\ 2,; ,, C V (‘\ NH) T ‘ Ll ~~>~l l NH or 1: a] +H / r . \If’; N R Aside Ii Bside NADH (reduced) (05" NADPH) 1.0 Absorbance o 03 .0 4:. P to In NADP+ this hydroxyl‘ group is esterified with phosphate. 220 240 260 280 300 320 340 360 380 F"? ‘3 ' 21f Wavelength (nm) the pentose phosphate pathway serves to reduce peroxides Warburg found that NADP+ could be reduced to NADPH in a reaction with glucose 6- phosphate, which yields 6-phosphoglucono-o-lactone. Osc "or x” GLuCoSE é-PHoSPHHTE ‘ ,4 “gm + ’DEHYDROGENHSE ”:37, + NADPH + hf1L Hot.” 0 +NADP ——___—.—_—____> "932? HCOH Hg, ’ Hi1 - chopog' HszOS' c- rHosPHoGLaCouoLnCToNE In the 1950s, two more enzymes of the pentose phosphate pathway were discovered. 6-phosphogluconolactonase adds water to 6—phosphoglucono-5- lactone to give 6-phosphogluconate, and phosphogluconate dehydrogenase causes reduction of another molecule of NADP“, with release of carbon dioxide and ribulose 5-phosphate. {3:1le O \ O— \ C / NADPH + 002 NADPH + H+ H {I} OH CH OH CHzopofi" NADP+ f CH20P03” H20 H+ HO (3: H NADP+ if (g: 2 H O OH \ A H 0 x — — \v :0 H \W H $34, * :xL, ‘ OH H W OH H O H~—C—OH M H—C'~OH HO H glucose- HO 6-phospho- I 6-ph03pho- ‘ 6-phosphate glucono- H'— $_ OH gluconate Hm." (I: _OH H OH dehydrogenase H OH lactonase CH20P03' dehydro- CHQOPOESZ‘ genase Glucose-G- 6-Phosphoglucono- 6-Phospho- Ribulose-E- phosphate (G6?) B-lactone gluconate phosphate (RuEP) H \ (I: éo transketolase H -— C - OH ribulose-5~phosphate H (I: ' OH isomerase 0§ /H 1 ' l , 2, (‘3 - H~—- (I: —~ OH CH20P03 H—(lj—OH + H—(Ij—-0H RiboseJi- , V _ _ _ phosphate (REP) CHZOPOE' CHZOPO§‘ Glyceraldehyde' sedohe'p’tulos'e-‘i- (‘H OH 3-phosghate phoSph’ate (S7P) f 2 (GAP) — '7 transaldolase transketolase FIGURE 23-25 The pentose phosphate pathway. The number of lines in an arrow represents the number of molecules react- ing in one turn of the pathway so as to convert three G6Ps to three C025, two F6135, and one GAP. For the sake of clarity, Sugars from Reaction 3 onward are shown in their linear forms Feat 11 ribulosevS-phosphate I epimerase Xylulos‘efis . phosphate (X11511) The carbon skeleton of RSP and the atoms derived from it are drawn in red and those from XuSP are drawn in green. The Q units transferred by transketolase are shaded in green and the C} units transferred by transaldolase are shaded in biue. GLMoNo— 0s 9mg; ME 0 /O'+H+ G'PHOSNOGWWME H (5:) HQ temmaewase “to“ (:0 0H ' VP: Hoe“ HocH 3 + H~éoH + H flg'ou H6054 NADP H' H + Col ,0 HcoH 5° 5 ’D-RiBuLose é-PHOWHOQWCONOLHCTONE 6— PHOSPHoGLMCONATE S-THOSPHHTE Thus, three enzymes constitute the oxidative part of this pathway. The activity of this pathway keeps the NADPH/NADP+ ratio at 1,000/1. In contrast, the NADH/NAD+ ratio is 1/ 100, so there is a hundred thousand fold difference in the oxidation states of the NAD+ and NADP+ pools. NADPH is always ready for biosynthesis, whereas NAD+ is always ready to carry hydrogens and electrons to the electron transport chain to make ATP and water. A genetic marker in the oxidative part of the pentose phosphate pathway The glucose 6-phosphate dehydrogenase isozyme expressed in red blood cells is sex-linked. In African America...
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