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SUBJECT: BIOCHEMISTRY TOPIC: CARBOHYDRATE
M ETABOLISM 1 (GLYCOLYSIS &
LECTURER: DR. UY DATE: DECEMBER 2010
# ▬ ↑ ↓
= Clinical correlation; = Step number (Glycolysis) = Positive regulation = Negative regulation = increase/d =decrease/d
Glucose: C 6 H 12 O 6 • Beginning or end of major pathways of carbohydrate metabolism • Major form in which carbohydrates absorbed form GIT is presented to cells • Major fuel for the brain • Biomedical importance o Provide ATP in absence of oxygen allowing tissues to survive anoxic episodes o Heart muscle is adapted to aerobic performance ↓ glycolytic pathway; poor survival under conditions of ischemia or myocardial infarction hemolytic anemia: pyruvate kinase deficiency fatigue: phosphofructokinase deficiency cancer cachexia: ↑ lactate; ↑ gluconeogenesis; hypermetabolism lactic acidosis: impaired activity of pyruvate dehydrogenase Relationship of Glucose to Major pathways of Carbohydrate Metabolism
3 Stages of Glycolysis I. Primary Stage
D-Glucose + 2ATP → D-fructose 1,6–Bisphosphate 2ADP + 2H +
A.k.a. Trapping stage – trapping of glucose in form of Glucose 6-Phosphate (G6P) with the utilization of ATP. Phosphorylation with a negatively charged PO4 to Glucose forming G6P, glucose is prevented from moving outside of the cell therefore “trapping” it inside the cell.
In von Gierke disease, aka Type I Glycogensosis (Glycogen storage disease) deficiency of Glucose6-Phosphatase (G6Pase) which catalyzes the hydrolysis of G6P to D-Glucose, preventing G6P to be converted to D-glucose which can be transported outside the cell. This results in the accumulation in the liver of excessive amounts of normal glycogen. II. Splitting Stage
D-fructose 1,6-Bisphosphate → 2 D-Glyceraldehyde 3Phosphate
(Harper’s Illustrated Biochemistry Chapter 18) •
Splitting of FBP into two Triose phosphates (G3P)
A.k.a. Embden-Meyerhoff Pathway A Catabolic Pathway (TCA = Amphibolic Pathway) Used by all cells to extract energy from glucose Aerobic or Anaerobic
III. Oxido-reductase Phosphorylation Stage 2D-G3P + 4ADP + 2Pi + 2H + → 2Lactate + 4ATP + 2H 2 O • Earning stage – formation of ATP *Stage I and II are the investment stage (Investment of 2 ATPs). *In the energy generation phase 4 ATP and 2NADH will produce 3ATP each = 6ATP in the ETC. *Glycolysis yields a net of only 2 ATP because the other 2
A) Aerobic glycolysis –Pyruvate will be oxidized to CO2, H2O, and Energy (ATP); B) Anaerobic glycolysis – Pyruvate is converted to Lactate utilizing NADH to NAD+ which is used by Glyceraldehyde to form 1,3bis-Phosphoglycerate.
Stage I and !! : Investment phase Stage III : Energy generation phase
GLUT4 – (in the muscles and adipose tissues) insulin dependent transporters In DM type 1 patients, lack of insulin causes fatigue due to lack of ATP caused by the lack of glucose transported inside. GLUT2 – (in liver and pancreas)
GLYCOLYSIS STEP 1 - Phosphorylation by Hexokinase/Glucokinase (Irreversible) - Trapping of glucose by phosphorylation to G6P DIFFERENCE BETWEEN HEXOKINASE GLUCOKINASE
*This is where the 1st ATP is invested.
GKRP attaches to glucokinase in its inactive form in the nucleus – forming a complex with GK.
With the increase in glucose concentration in the cell, release of GK by GKRP into the cytosol is promoted – converting GK to its active form. While the increase of Fructose 6-Phosphate (F6P) signals the inactivation of GK back into the nucleus with GKRP therefore inhibiting its activity. M ATURITY -O NSET D IABETES OF THE Y OUNG (MODY) Type 2 An autosomal dominant disorder involving mutations in the glucokinase (GCK) gene.
Hexokinase: Low Km for glucose relative to its concentration in blood. (high affinity for glucose) Abundant in the Muscles o Hexokinase immediately reacts even with a low concentration of glucose. o This rapid reaction of the enzyme is important for G6P to be readily available in the muscles. Strongly inhibited by its product Glucose 6Phoshate (G6P) hence its reaction is not at equilibrium. o When there is enough G6P in the muscle Hexokinase is inhibited. (Low Vmax) In liver, hexokinase is saturated under normal condition Glucokinase (GK): S 0.5 for glucose is higher than the Km for glucose of other kinases less sensitive to product inhibition by G6P
(G6P does not inhibit Glucokinase)
Patients have progressive hyperglycemia that is usually asymptomatic at diagnosis and is usually managed with diet alone. STEP 2 – Isomerisation by Phosphoglucose isomerise (Reversible)
contribute to the capacity of the liver to “buffer” blood glucose levels because of the following features o When there is too much glucose it will be released. A. High (Km) S 0.5 for glucose
STEP 3 – 2 nd Phosphorylation by PFK-1 (Rate Limiting Step)
is decreased which causes a decrease in (↓) cAMP and decreasing the activation of (↓) PKA which causes increased (↑) dephosphorylation of a bifunctional enzyme Phosphofructokinase2(PFK-2)/Fructose 1,6-Bisphosphatase2(FBP-2) complex (active PFK-2; inactive FBP-2). PFK-2 when active converts F6P into Fructose 2,6Bisphosphate which increases activity of PFK-1. . ∴↑Glucose = ↑ Glycolytic activity o On the opposite effect... (not shown in the diagram) During starvation, The bifunctional enzyme is phosphorylated deactivating PFK-2 and activating FBP-2 which converts Fructose 1,6bisphosphate to F6P (a reverse of the function of PFK-1); decreasing the activity of PFK-1.∴↓Glucose = ↓ Glycolytic activity
PHOSPHOFRUCTOKINASE (PFK-1) DEFICIENCY
During this step the 2nd ATP is invested; And F6P is phosphorylated with Phosphofructokinase-1 (PFK1) into Fructose 1,6-bisphosphate (FBP). Regulatory step / Rate limiting step / 1st committed step of glycolysis. ▬ Abundance of ATP and Citrate regulates glycolysis at this step by inhibiting the activity of PFK-1.
A form of glycogen storage disease or Glycogenosis (Type VII) *Defined: Glycogenosis - Any of the (12 types of) glycogen deposition diseases characterized by accumulation of glycogen of normal or abnormal chemical structure in tissue Normal glycogen accumulates in muscles Results in inefficient use of glucose stores by RBCs and muscles Patients experience hemolytic anemia and muscle cramping
Increased amount of AMP occurs during starvation which means there is a high demand of ATP therefore promoting the activity of PFK-1. Fructose 2,6-bisphosphate (FBP) increases during fed state which promotes PFK-1 activity.
(See Diagram below and explanation continued for FBP.)
*’bis-‘ indicates phosphate groups are separated, whereas ‘di-‘ indicates that the phosphate groups are joined e.i. ADP. STEP 4 – Cleavage by Aldolase
A.k.a. the splitting stage as previously mentioned in page 1
ALDOLASE A ( found in RBCs and muscle) Absence of the enzyme (aldolase) may cause nonspherocytic hemolytic anemia. *most/almost all of glycolytic enzyme deficiencies manifest hemolytic anemia because this causes the ‘pumps’ in the membrane of RBC do not function properly. This allows the RBC to be exposed to oxidative properties which causes hemolysis or swelling of the RBC. *In this disease however the hemolytic anemia is nonspherocytic which means the RBCs are not spherical or sphere-shaped like most hemolytic anemia. TRIOSE PHOSPHATE ISOMERASE (TPI) Patients with TPI deficiency have neonatal hemolytic anemia and progressive neurologic involvement Progressive hypotonia with eventual diaphragm paralysis and cardiomyopathy.
STEP 5 – Most DHAP are converted to G3P which is utilized in the glycolytic pathway.
x 2 9 4
*because most DHAP is converted to G3P there should be 2 molecules of G3P converted to Pyruvate.
STEP 6 – oxidation by G3P dehydrogenase As previously mentioned most of the DHAP will be converted to G3P, which G3P is the one utilized in the formation of Pyruvate, therefore from everything beginning in step 5 happens in pair. In this step, G3P is converted to a high energy compound 1,3x
Bisphosphoglycerate (1,3BPG) by Glyceraldehyde 3-phosphate dehydrogenase (G3PDH or GAPDH) the reduction of NAD + to NADH+H + which will form (3)ATP* in the ETC or utilized by the reduction of pyruvate to lactate catalyzed by lactate dehydrogenase in anaerobic glycolysis. *2(NADH+H+)
therefore, 6 ATPs
STEP 7 – 1 st substrate level Phosphorylation by Phosphoglycerate kinase (PGK) (and ADP ATP) and formation of 2,3BPG byproduct. At this step, 1,3BPG is converted to 3-Phosphoglycerate by transferring of the phosphate to ADP forming ATP. This is the first substrate level phosphorylation in Glycolysis. 2ATPs should be formed because there are 2 molecules of 1,3BPG from the splitting. At this point of glycolysis the 2ATPs invested in steps 1 and 3 has already been regained. There is also the presence of an enzyme mutase, abundant in the RBC, which may form a byproduct 2,3BPG from 1,3BPG. 2,3BPG shifts the oxygen dissociation curve to the right – releasing more oxygen to the tissues. 2,3BPG can then be converted to 3-Phosphoglycerate(3PG) with the use of the enzyme Phosphatase. *2,3BPG or diphosphyglycerate/DPG is also likely an intermediate of the reaction. *However, this pathway does not produce ATP. *PGK is inhibited by arsenate Arsenic toxicity – arsenate competes with the inorganic phosphate (Pi) hydrolyzing 3PG to 2PG without ATP formation STEP 8 mutase – Isomerization by Phosphoglycerate
During starvation, glucagon is increased. This stimulates the activity of adenylyl cyclase which converts ATP to cAMP. cAMP then activates PKA. PKA phosphorylates Pyruvate kinase(PK) turning it into its inactive form. Therefore, with decreased glucose glycolysis decrease production of pyruvate with the inactivation of PK.
*kinases are inactive when phosphorylated similar to PFK-2
During Anaerobic glycolysis pyruvate forms Lactate catalyzed by the enzyme Lactate dehydrogenase utilizing with it. NADH+H+ from the activity of G3P dehydrogenase
3-Phosphoglycerate is isomerized to 2-Phosphoglycerate STEP 9 – Dehydration catalyzed by Enolase 2-Phosphoglycerate removes H2O to form a high energy phosphate compound phosphoenol pyruvate(PEP) *enol compounds=high energy compounds Enolase is inhibited by Flouride; used to inhibit glycolysis in blood samples used for measuring glucose. STEP 10 – 2 nd Substrate level phosphorylation (Irreversible) At this step, phosphate is transferred from PEP to ADP by pyruvate kinase(PK) – forming 2 molecules of ATP per glucose. Since the invested ATPs were already regained, this 2nd substrate level phosphorylation is the first gaining of ATP from glucose. PK is allosterically activated by FBP(product of the rate limiting step – page2) Pyruvate Anemia Kinase Deficiency & Hemolytic
2 ATP consumption with the enzymes Hexokinase/Glu cokinase and Phosphofructoki nase-1(PFK-1). 2NADH+2H + produced from G3P dehydrogenase.
Lack of ATP affects ion pumps especially Na+/K+ ATPase. –causing the cells to swell & lyse
4ATP produced from (2)Phosphoglycerate kinase (PGK) and (2) (PK Pyruvate kinase) In anaerobic pathway, 2 NADH from G3PDH is utilized to convert
3 I RREVERSIBLE STEPS OF GLYCOLYSIS WHICH ARE ALSO
which is also permeable to the inner mitochondrial membrane then exits the mitochondria and converted back to OAA by AST. Fate of the PYRUVATE
Carboxylation of Pyruvate Oxaloacetate (OAA) is catalyzed by Pyruvate carboxylase.
*All are kinase enzymes. NADH Generated by Glycolysis has to be oxidized back to NAD+. This is done by the enzyme Lactate Dehydrogenase(anaerobic) & Substrate Shuttles(aerobic). In Aerobic glycolysis, NADH produced by G3PDH needs to be transported into the mitochondria for ATP synthesis in the ETC, however, NADH is NOT permeable to the inner mitochondrial membrane. By utilizing substrates (Malate and Aspartate) permeable to the inner mitochondrial membrane, NADH is made available to the ETC in the mitochondria. OAA + NADH →Malate Dehydrogenase→ Malate + NAD+
(Oxidative) Decarboxylation of Pyruvate to Acetyl CoA is catalyzed be Pyruvate deydrogenase complex along with the reduction of NAD+. Acetyl CoA will proceed to TCA cycle. The Pyruvate dehydrogenase complex is a multienzyme complex which consists of Pyruvate Dehydrogenase, Dihydrolypoyl Tranacetylase, Dihydrolipoyl Dehydrogenase.
(The succeeding discussion on Oxidative decarboxylation was not discussed but it was in the PPT and is also found in the Harpers on chapter 18, page 153, “The oxidation of pyruvate to Acetyl-CoA is the irreversible route from glycolysis to the citric acid cycle”.)
During the conversion the intermediates do not dissociate and remain bound to the components of the multienzyme complex. MECHANISM: (OXIDATIVE CARBOXYLATION OF PYRUVATE) Pyruvate is decarboxylated to a hydroxyethyl (Acyl) bound to thiamine diphosphate (TPP or TDP) with the enzryme Pyruvate dehydrogenase, forming Acyl-TPP. Acyl-TPP reacts with an oxidized lipoamide (Lip-S2) catalyzed be the Dihydrolipoyl Transacetylase component, forming AcylLipoate/Acetyl lipoamide (in Harper’s). Acyl-lipoate will then reacts with Coenzyme A(CoA-SH) forming Acetyl CoA and the reduced lipoamide. However lipoamide has to be returned to its original form which is reoxidized by the Dihydrolipoyl Dehydrogenase(flavoprotein component containing an FAD, forming FADH and Lip-S2. Dihydrolipoyl Dehydrogenase is finally oxidized by NAD+ for it to return to its orginal form – flavoprotein containing FAD.
NADH is oxidized back to NAD+ with Malate dehydrogenase (MDH) reducing Oxaloacetate OAA to Malate which is permeable to the inner mitochondrial
Reaction Catalyzed by Glyceraldehyde 3phosphate dehydrogenase Phosphoglycerate kinase Pyruvate kinase
Method of ATP Formation Respiratory chain oxidation of 2 NADH Substrate level phosphorylation Substrate level phosphorylation
ATP per Mol of Glucose 6*
GLUCONEOGENESIS (Harper’s Illustrated Biochemistry Chapter
2 2 10
Consumption of ATP for reactions of hexokinase and phosphofructokinase
–2 Net 8*
Defined: The net synthesis or formation of glucose from non-carbohydrate substrates: - Amino acids (Except lysine & leucine) - Lactate - Pyruvate - Propionate - Glycerol - Fructose Essential for survival of humans and other animals
Citric acid cycle
Pyruvate dehydrogenase Isocitrate dehydrogenase -Ketoglutarate dehydrogenase Succinate thiokinase Succinate dehydrogenase Malate dehydrogenase
Respiratory chain oxidation of 2 NADH Respiratory chain oxidation of 2 NADH Respiratory chain oxidation of 2 NADH Substrate level phosphorylation Respiratory chain oxidation of 2 FADH2 Respiratory chain oxidation of 2 NADH
6* 6* 6* 2
Enzymes Unique To Gluconeogenesis (Irreversible reactions) (The rest may be found in Glycolysis) • Mitochondrial: 1. Pyruvate Carboxylase – cannot act in the cytosol Pyruvate→OAA • Cytosolic: 2. PEP Carboxykinase (PEPCK) OAA →PEP 3. Fructose-1,6-Bisphosphatase (FBPase) FBP → F6P 4. Glucose-6-Phosphatase (G6Pase) G6P → Glucose
You may refer to Harper’s Ill. Biochem: Figure 20-1 for a more detailed figure.
4 6 Net 30
Total per mol of glucose under aerobic conditions Total per mol of glucose under anaerobic conditions
(Values are changed assuming 2ATPs from FADH and 3 from NADH)
6-Phosphofructo-1-kinase is a Regulatory Enzyme of Glycolysis ▬ Negative Allosteric Effectors 1. Citrate 2. ATP 3. Hydrogen ions (low pH) Positive Allosteric Effectors 1. AMP 2. Fructose 2,6 – bisphosphate Rate of Glycolysis Depends On: Energy state of the cell (ATP or AMP) Internal environment of the cell (H ions) ↑H+=acidosis Availability of alternate fuels such as fatty acids and ketone bodies (citrate) Insulin / glucagon ratio in the blood (F - 2,6 – BP) Effect on Rate ↑ATP = ▬ ↑AMP = ↑H+= ▬ ↑Citrate = ▬ ↑Insulin/Glucagon Ratio = Remember: -Aerobic -Anaerobic leading to Lactic Acidosis -Regulatory steps -Mutations
(For the Glycolysis summary please refer to slide 49 of the PPT)
Lactate as substrate for gluconeogenesis
2Lactate + 6ATP + 6H2O → Glucose + 6ADP + 6Pi + 4H+ Glucose can be immediately trapped by the muscle with the enzyme Hexokinase. During exercise there will be an increased amount of lactate causing lactic acidosis. This will cause pain in the muscles (commonly in the form of cramps). Because of muscles do not have lactate dehydrogenase Lactate will be transported to the liver where it can be converted to glucose. In the liver lactate dehydrogenase can catalyze the oxidation of Lactate to Pyruvate which requires 6ATP to form glucose (gluconeogenesis) to be sent back to the muscle and other tissues. Utilizing 6ATP makes gluconeogenesis an ‘expensive’
Carboxylation of Pyruvate is catalyzed by Pyruvate carboxylase by transferring the CO2 attached to biotin forming Oxaloacetate (OAA). Because OAA is not permeable to the mitochondrial membrane it is reduced by NADH to malate. In the cytosol, malate is reoxidized to OAA with NAD+. OAA will then be converted (decarboxylation and phosphorylation) to Phosphoenol pyruvate (PEP) by PEP carboxykinase (PEPCK) with the cofactor GTP(→GDP). The succeeding steps beginning from PEP are reverse reactions of glycolysis, except in FBPase & G6Pase. ATP is again utilized in the formation of 1,3-BPG. Because 2 molecules is required in the formation of FBP 2 pyruvate must be utilized to form 1 molecule of glucose. After this step, 4 ATP and 2GTP has been utilized by the pathway. 2NADH will be utilized to convert 2 molecules of1,3BPG to form 2 molecules of G3P. PFK-1 is an irreversible enzyme of glycolysis therefore gluconeogenesis needs its own enzyme FBPase. The last step of gluconeogenesis is catalyzed by Glucose6-phosphatase(G6Pase) which dephosphorylates G6P to Glucose. G6P is not found in the muscles therefore, gluconeogenesis cannot occur in the muscle von Gierke’s Disease (mentioned in page 1) type I glycogen storage disease(GSD) deficiency of glucose-6-phosphatase severe hypoglycemia causing lethargy,seizures and brain damage hepatomegaly, increased bleeding (clotting factors 2,6,7,9,10 and platelets are affected) and growth retardation Glucagon Main gluconeogenic hormone Similar to the previous discussion in page 3 and 5, attachment of the hormone glucagon deactivates PK
Amino acids as substrate for Gluconeogenesis In the muscles Alanine transaminase or Alanine aminotransferase (ALT) catalyze the transferring of an amino group from glutamate to pyruvate forming αketoglutarate and alanine. Through the blood Alanine travels to the liver where it is converted back to pyruvate with ALT. Aside from the 6 ATP required for pyruvate to undergo gluconeogenesis, Ammonium needs to be released from glutamate through urea cycle utilizing 4 ATP. This makes amino acids as substrate more expensive (ATP). 2Ala + 10ATP + CO2 → Glucose + Urea + 10ADP + 10Pi
Excessive amounts of ammonia loaded into the liver, up to a point that not everything can be immediately converted to urea can cause comatose due to hepatic encephalopathy. This is common in liver cirrhosis caused by alcoholism. GLUCOKINASE IN THE LIVER • • Regulates blood glucose after a meal Promotes increased hepatic utilization of glucose o Promotes Glycolysis not gluconeogenesis nor glycogenolysis.
↑Catecholamine increase secretion of Glucagon which is synthesized from α-pancreatic cells.
During starvation a great amount of ATP is used for gluconeogenesis which may be taken from the oxidation of fatty acids in the liver. This process however forms ketone bodies. Insulin
G LUCOSE IS S YNTHESIZED FROM M OST A MINO A CIDS • All amino acids except leucine & lysine can supply carbon for the net synthesis of glucose by gluconeogenesis Pyruvate, oxaloacetate – catabolic products of amino acids from which glucose synthesis can occur Anaplerotic Reactions – those that lead to net synthesis of TCA intermediates
• the only counter-regulatory hormone in gluconeogenesis • main glycolytic hormone • action o binds to receptor o receptor sends signal activating phosphoprotein phosphatase o inactive PFK-2 becomes active PFK-2 (Pi removed) o active PFK-2 increases concentration of F-2,6 BP o F-2,6 BP binds to PFK-1 & increases its activity o Rate of glycolysis is increased Alcohol Oxidation Inhibits Gluconeogenesis Due to excess NADH forcing the equilibrium of the reactions catalyzed by lactate dehydrogenase and malate dehydrogenase → PEPCK Deficiency rare but severe metabolic defect absence of the cytosolic form results in cerebral atrophy, optic atrophy, fatty infiltration of the liver and kidney and intractable hypoglycemia (including lost of vision) Presents with neonatal hypoglycemia, along with acidosis, irritability, tachycardia, dyspnea, hypotonia, moderate hepatomegaly. typically only the liver enzyme is deficient, the muscle activity is normal
Hepatic encephalopathy – utilization of amino acids for gluconeogenesis may cause production of excess urea which may lead to an encephalopathy associated with cirrhosis of the liver, attributed to the passage of toxic nitrogenous substances from the portal to the systemic circulation; cerebral manifestations may include coma. Gluconeogenic Glycine Serine Valine Histidine Arginine Cysteine Proline Hydrodyproline Alanine Glutamate Glutamine Aspartate Asparagines Methionine Ketogenic Leucine Lysine Both Threonine Isoleucine Phenylalanine Tyrosine Tryptophan
Fructose-1,6-Bisphosphatase Deficiency -
(Please refer to slide 82 of the PPT for the gluconeogenesis summary)↓
Glucose can be synthesized from Propionyl-CoA (odd-numbered carbon) • • Propionyl-CoA – good precursor for gluconeogenesis as it yields oxaloacetate by anaplerotic pathway(TCA cycle). Triacylglycerol(TAG) – when hydrolyzed yields 3 FA’s & glycerol (a substrate for gluconeogenesis) o Because Glycerol kinase(found in adipose tissue) is absent in the liver, TAG needs to be hydrolyzed to FAs and glycerol before becoming a substrate for gluconeogenesis.
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