Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a...
Transcript of Glycolysis & Gluconeogenesis 1 - Web Publishing · Each molecule of the glycolytic pathway has a...
Glycolysis & Gluconeogenesis 1
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
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.
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?�
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? �
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. �
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. �
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.�
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.
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.
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. �
Hexokinase I, II & III
Glucokinase: Hexokinase IV
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.
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.�
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.
• 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.
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).
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.
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.
Fructose 1,6 PO4/triose PO4 lyase; Aldolase
+
Fr 1,6 BP GAP DHAP
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 �
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.�
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
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.
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.�
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).�
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.
. �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.�
• 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.
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
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.
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.
Transport of PEP and oxaloacetate
from the mitochondrion to
the cytosol.
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.