Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a...

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Glycolysis & Gluconeogenesis 1

Transcript of Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a...

Page 1: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Glycolysis & Gluconeogenesis 1

Page 2: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

FORW

ARD

 REACTION  

REVERSE  REACTION  

ΔGo  (kJ)   K   Significance  

200  

100  

50  

10  

1  

0  

-­‐1  

-­‐10  

-­‐50  

-­‐100  

-­‐200  

9x10-­‐36  

3x10-­‐18  

2x10-­‐9  

2x10-­‐2  

7x10-­‐1  

1  

1.5  

5x101  

6x108  

3x1017  

1x1035  

EssenJally  no  forward  reacJon;  reverse  reacJon  goes  to  compleJon  

Forward  and  reverse  reacJons  proceed  to  same  extent  

Forward  reacJon  goes  to  compleJon;  essenJally  no  reverse  reacJon  

ΔG = RT ln Q/K = RT lnQ - RT ln K Under standard conditions (1 M concentrations, 1 atm for gases), Q = 1 and ln Q = 0 so ΔGo = - RT ln K  

Page 3: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Factors affecting the activity of enzymes. The total activity of an enzyme can be changed by altering the number of its molecules in the cell, or its effective activity in a subcellular compartment (1 through 6), or by modulating the activity of existing molecules (7 through 10). An enzyme may be influenced by a combination of such factors.

Page 4: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on
Page 5: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on this chart. �What is the significance of this?�

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These are the glucose transporter found in a typical mammalian cell. They are found in different cell types found in different tissues. There are other, such as the Na+ Glucose transporter of the intestinal cells. Why are there so many different transporter? Consider if you a measuring glucose utilization by a specific cell type, should the kinetics of transport be considered? �

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There are several fates for glucose when it enters an cell; �

•  These fates are all following the initial rxn that forms glucose 6-P04.�

•  The pathways that claim G6P are; pentose phosphate pathway, glycogen synthesis pathway as well as entering glycolysis. �

•  The final product of glycolysis is pyruvate. In mammalian cells it has two major fates, homolactate fermentation or aerobic respiration. �

Page 8: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Glycolysis can be though of as consisting of three separate portions.�

•  Stage 1 is the phosphorylation of glucose and the isomerization to Fr-6-P04; why is this step necessary?�

•  Stage 2 is the isomerization of GAP and DHAP. Why is DHAP the favored product? �

•  Stage 3 is the utilization of GAP to form pyruvate. �

Page 9: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Glucose + 2NAD 2pyruvate + NADH �

ΔG0’ = -146 kJ/mol�2ADP + 2Pi 2ATP + H2O�ΔG0’ = 61 kJ/mol�Overall free energy -85 kJ/mol�Glycolysis is not a reversible�pathway.�Gluconeogensis then must be a

separate pathway.�Glucose2 Lactate �ΔG0= -196 kJ/mol�But the lactate dh is reversible,

there are 5 isoenzymes of Ldh in most mammals.�

Page 10: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on
Page 11: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Why are gluconeogensis and glycolysis are

reciprocal pathways? •  Three major enzymes

are not reversible due to their thermodymanics, these are;

•  Hexokinase •  PFK-1 •  Pyruvate kinase. •  We will return to this

slide later.

Page 12: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Gluconeogenesis is a reciprocal pathway to glycolysis, and not a mere reversal of glycolysis. At specific highly exothermic reaction in glycolysis, the reverse reaction is not possible. A second enzyme is necessary that reverses that reaction step. There are three steps of this kind, Pyruvate Kinase, PFK-1/PFK-2, and Hexokinase/Glucokinase. Non- carbohydrate precursors of glucose via gluconeogenesis are lactate, pyruvate, amino acids (primarily alanine) and Krebs cycle intermediates. The only amino acid carbon chains not available for gluconeogenesis are lys & leu. There product is acetyl-CoA.

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Comparison  of  the  rela0ve  enzyma0c  ac0vi0es  of  hexokinase  and  glucokinase  over  the  physiological  blood  glucose  range.    KM for this enzyme is ≈5mM with a Hill constant of 1.5.�It activity increases rapidly over the physiological [glucose]. Glucokinase is not inhibited by physiological [G6P]. Liver cells have GLUT 2, which is not dependent on insulin for expression, (GLUT 4 is insulin dependent). Thus the liver does not compete with other tissues for blood glucose. When [glucose] is low, it supplies glucose, and when [glucose] is high liver converts glucose to G6P. The high G6P allosterically facilitates glycogen synthesis and inactivates glycogen phosphorylase & PP1. �

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Hexokinase  I,  II  &  III  

Glucokinase:  Hexokinase  IV  

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Regulation of hexokinase IV (glucokinase) by sequestration in the nucleus. The protein inhibitor of hexokinase IV is a nuclear binding protein that draws hexokinase IV into the nucleus when the fructose 6-phosphate concentration in liver is high and releases it to the cytosol when the glucose concentration is high.

Page 16: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

         Hexokinase reaction is a model for all kinases in glycolysis PFK, glyceraldhyde kinase, pyruvate kinase.�

Explain the ‘clam shell model’, the importance of excluding water from the active site, and the role of Mg+2.�

Page 17: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Glucose-6-phosphate isomerase (alternatively known as phosphoglucose isomerase or phosphohexose isomerase) is an enzyme that catalyzes the conversion of glucose-6-phosphate into fructose 6-phosphate in the second step of glycolysis.

PGI monomers are made of two domains, one made of two separate segments called the large domain and the other made of the segment in between called the small domain. The two domains are each αβα sandwiches, with the small domain containing a five-strand β-sheet surrounded by α-helices while the large domain has a six-stranded β-sheet. The large domain and the C-terminal of each monomer also contain "arm-like" protrusions. Functional PGI is a homodimer.

Page 18: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

•  This is the committed step to glycolysis, the product F16BP is the only molecule that can be cleaved by the reverse Classen condensation rxn, by aldolase A to form GAP + DHAP.  

•  PFK-1 activity is controlled by the product of PFK-2, the homotropic activator F26BP.

Page 19: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

ROLE OF FRUCTOSE 2,6-BISPHOSPHATE IN REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS. FRUCTOSE 2,6-BISPHOSPHATE (F26BP) HAS OPPOSITE EFFECTS ON THE ENZYMATIC ACTIVITIES OF PHOSPHOFRUCTOKINASE-1 (PFK-1, A GLYCOLYTIC ENZYME) AND FRUCTOSE 1,6-BISPHOSPHATASE (FBPASE-1, A GLUCONEOGENIC ENZYME).

Page 20: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Role of fructose 2,6-bisphosphate in regulation of glycolysis and gluconeogenesis. Fructose 2,6-bisphosphate (F26BP) has opposite effects on the enzymatic activities of phosphofructokinase-1 (PFK-1, a glycolytic enzyme) and fructose 1,6-bisphosphatase (FBPase-1, a gluconeogenic enzyme).

FBPase-1 activity is inhibited by as little as 1 µM F26BP and is strongly inhibited by 25 µM. In the absence of this inhibitor (blue curve) the K0.5 for fructose 1,6-bisphosphate is 5 µM, but in the presence of 25 µM F26BP (red curve) the K0.5 is >70 µM. Fructose 2,6-bisphosphate also makes FBPase-1 more sensitive to inhibition by another allosteric regulator, AMP.

Page 21: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Forma&on  and  degrada&on  of  β-­‐D-­‐fructose-­‐2,6-­‐bisphosphate  as  catalyzed  by  PFK-­‐2  and  FBPase-­‐2.    F2,6BP  is  a  potent  allosteric  ac=vator  of  PFK-­‐1,  and  inhibitor  of  F1,6  BPase.    It  is  not  a  glycoly=c  metabolite.    It  synthesis  is  dependent  on  PFK-­‐2  &  F2,6  BPase.    PFK-­‐2/F2,6Pase  are  two  different  ac=ve  sites  on  100kD  homodimeric  protein.    Each  subunit  has  both  ac=ve  sites.    The  enzyme  is  subjected  to  allosteric  regula=on  as  well  as  PO4  by  PKA.    PO4  at  ser  32  lead  to  inhibi=on  of  PFK-­‐2  ac=vity  and  ac=va=on  of  F2,6BPase.    Pancrea=c  α  cells  release  glucagon  in  response  to  low  blood  [glucose]  resul=ng  in  an  increase  in  liver  [cAMP],  decrease  in  F2,6BP,  decrease  in  PFK-­‐1  ac=vity,  inhibi=ng  glycolysis,  deinhibi=on  of  FBPase-­‐1,  s=mula=ng  gluconeogenesis.    When  blood  [glucose]  is  high,  [cAMP]  in  liver  decreases,  PFK-­‐2/FBPase-­‐2  in  dephosphorylated  by  PP1,  ac=va=ng  the  PFK-­‐2  ac=vity,  causing  an  increase  in  F2,6BP,  which  then  ac=vates  PFK-­‐1  and  FBPase-­‐1  inhibited.      

Page 22: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

Fructose 1,6 PO4/triose PO4 lyase; Aldolase

+  

Fr 1,6 BP GAP DHAP

Page 23: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

What is the limiting reagent of glycolysis?�•  NADH is generated by the

oxidation of GAP in the formation of 1,3DPG. This is an endothermic rxn.    

•  What drives it then to occur? �

•  DHAP + NADH Glycerol 3 PO4 + NAD �

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Homolactate fermentation is not the only way to generate NAD from reduced NADH. This is a very important pathway. This pathway generates ATP and produces glycerol for triacylglyceride synthesis in the liver. �

The glycerophosphate dh is a transmembrane protein found in the outer surface of the inner mitochondrial membrane, it ultimately transfers the H+ from the cytosolic NADH to a mitochondrial FAD to enter the ETS at complex 2, generating 2 additional ATP by aerobic respiration.�

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The glycerol-3-PO4 Shuttle

•  the enzyme called cytoplasmic glycerol-3-phosphate dehydrogenase (cGPdh) converts DHAP to glycerol 3-phosphate by oxidizing one molecule of NADH to NAD+

•  Glycerol-3-phosphate gets converted back to DHAP by a membrane-bound mGPdh, this time reducing one molecule of enzyme-bound FAD to FADH2. FADH2 then reduces coenzyme Q (ubiquinone to ubiquinol) which enters into oxidative phosphorylation. This reaction is irreversible

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TIM  is  a  dimer  of  iden=cal  subunit  of  250  aa  each,  he  three-­‐dimensional  structure  of  a  subunit  contains  eight  α  helical  regions  outside  of  eight  β  parallel  sheet  regions.  This  structural  mo=f  is  called  an  αβ-­‐barrel,  or  a  TIM  barrel,  the  ac=ve  site  is  inside  the  barrel.    

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This reaction only works because the first reaction by GAPdh (+6.3 kJ/ mol) is driven by the second reaction of GK (-18.8kJ/mol). These reactions are coupled as is a pair of proteins that associate with one another.�

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The  Pyruvate  Kinase  rxn  

•  This is a very exothermic rxn , -31.4 kcal/mol�

•  It generates an ATP and forms pyruvarte. �

•  For gluconoegenesis to be turned on this rxn must be turned off, WHY?�

•  The major sources of pyruvate are red blood cells, they are amitochondail, and muscle tissue that are much more anaerobic as they become more active than utilizing pyruvate by aerobic respiration. The liver back converts pyruvate (formed from lactate & alanine).�

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Regulation of PK. The enzyme is allosterically inhibited by ATP, acetyl-CoA, and long-chain �fa (all signs of an abundant energy supply), and the accumulation of fr 1,6-BP triggers its activation. Accumulation of alanine, which can be synthesized from pyruvate in one step, allosterically inhibits PK, slowing the production of pyruvate by glycolysis. The liver isozyme (L form) is also regulated hormonally. Glucagon activates cAMP-dependent protein kinase (PKA), which phosphorylates the PK L isozyme, inactivating it. When the glucagon level drops, a protein phosphatase (PP) dephosphorylates PK, activating it. This mechanism prevents the liver from consuming glucose by glycolysis when blood glucose is low; instead, the liver exports glucose. The muscle isozyme (M form) is not affected by this phosphorylation mechanism.

Page 30: Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a specific free energy. Note where ATP is in relationship to every other molecule on

. �Two alternative fates for pyruvate�Pyruvate can be converted to glucose via gluconeogenesis or oxidized to acetyl-CoA for energy production. The first enzyme in each path is regulated allosterically; acetyl-CoA, produced either by fatty acid oxidation or by the pyruvate dehydrogenase complex, stimulates pyruvate carboxylase and inhibits pyruvate dehydrogenase.�Pyruvate carboxylase functions in lipogensis as well as gluconeogenesis dependent on cellular metabolic control.�

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•  The Cori cycle invloves the utilization of lactate, produced by glycolysis in non-hepatic tissues, (such as muscle and erythrocytes) as a carbon source for hepatic gluconeogenesis. In this way the liver can convert the anaerobic byproduct of glycolysis, lactate, back into more glucose for reuse by non-hepatic tissues. Note that the gluconeogenic leg of the cycle (on its own) is a net consumer of energy, costing the body 4 moles of ATP more than are produced during glycolysis. Therefore, the cycle cannot be sustained indefinitely.

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Role of biotin in the pyruvate carboxylase reaction. The cofactor biotin is covalently attached to the enzyme through an amide linkage to the ε-amino group of a Lys residue, forming a biotinyl-enzyme. The reaction occurs in two phases, which occur at two different sites in the enzyme. At catalytic site 1, bicarbonate ion is converted to CO2 at the expense of ATP. Then CO2 reacts with biotin, forming carboxybiotinyl-enzyme. The long arm composed of biotin and the Lys side chain to which it is attached then carry the CO2 of carboxybiotinyl-enzyme to catalytic site 2 on the enzyme surface, where CO2 is released and reacts with the pyruvate, forming oxaloacetate and regenerating the biotinyl-enzyme. The general role of flexible arms in carrying reaction intermediates between enzyme active sites. Similar mechanisms occur in other biotin-dependent carboxylation reactions, such as those catalyzed by propionyl-CoA carboxylase and acetyl-CoA carboxylase

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Mechanism  of  gene  regula&on  by  the  transcrip&on  factor  FOXO1.  Insulin  ac=vates  the  signaling  leading  to  ac=va=on  of  protein  kinase  B  (PKB).  FOXO1  in  the  cytosol  is  phosphorylated  by  PKB,  and  the  phosphorylated  transcrip=on  factor  is  tagged  by  the  a`achment  of  ubiqui=n  for  degrada=on  by  proteasomes.  FOXO1  that  remains  unphosphorylated  or  is  dephosphorylated  can  enter  the  nucleus,  bind  to  a  response  element,  and  trigger  transcrip=on  of  the  associated  genes.  Insulin  therefore  has  the  effect  of  turning  off  the  expression  of  these  genes,  which  include  PEP  carboxykinase  and  glucose  6-­‐phosphatase.  

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The PEP carboxykinase promoter region, showing the complexity of regulatory input to this gene. This diagram shows the transcription factors (smaller icons, bound to the DNA) known to regulate the transcription of the PEP carboxykinase gene. The extent to which this gene is expressed depends on the combined input affecting all of these factors, which can reflect the availability of nutrients, blood glucose level, and other factors that go into making up the cell's need for this enzyme at this particular time. P1, P2, P3I, P3II, and P4 are protein binding sites identified by DNase I footprinting. The TATA box is the assembly point for the RNA polymerase II (Pol II) transcription complex.

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Transport  of  PEP  and  oxaloacetate  

from  the  mitochondrion  to  

the  cytosol.  

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The participation of two second messenger systems: the cAMP-mediated stimulation of glycogenolysis and inhibition of glycogen synthesis triggered by glucagon and beta-andrenoreceptor activation; and the IP3, DAG, and Ca2+-mediated stimulation of glycogenolysis and inhibition of glycogen synthesis triggered by alpha-adrenoreceptor activation. IP3 stimulates the release of Ca2+ from the endoplasmic, whereas DAG, together with Ca2+, activates protein kinase C(PKC) to phosphorylate and thereby inactivate glycogen synthase. G6Pase occupies the endoplasmic reticulum. Consequently, the cytosolically produced G6P is transported into the endoplasmic reticulum via the T1 G6P translocase, where it is hydrolyzed to glucose and Pi. The glucose and Pi are then returned to the cytosol by the T2 and T3 transporters, respectively, and the glucose is exported from the cell via the GLUT2 glucose transporter.

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