Dissection of a Metabolic Pathway

DISSECTION OF A METABOLIC PATHWAY

I. Energetically unfavorable steps in a metabolic pathway can often be overcome by biochemical pushing and pulling. For A + B C + D

A. ΔG = ΔGo + 2.3RT log [(C)(D)/(A)(B)]

For a single reaction:

1. If the concentration of products increases relative to reactants, what happens to ΔG?

2. If the concentration of products decreases relative to reactants, what happens to ΔG?

B. For a pathway the overall ΔG = Sum of the Individual ΔG values. C. Biologically irreversible steps: those where the ΔG is so negative (generally -6 kcal/mole or more negative in the forward direction) that the reverse reaction cannot occur because the energetic roadblock is too high: When these occur, they are usually involve utilization of high energy phosphate compounds (you must know material in table on p. 8 of Organic Chem handout) or oxidation reactions.

II. Deciphering what an enzyme does from its name.

A. Each rxn. you encounter will generally be catalyzed by a different specific enzyme B. Mutations in the amino acid sequence of the enzyme will often lead to a congenital

metabolic or structural abnormality. C. Most of the time, an enzyme’s name has two parts: the name of the substrate it

works on and the type of reaction it carries out: e.g., lactate dehydrogenase D. Some common types of reactions carried out by enzymes (fill in the blanks as you

encounter them in class):

Lecturer - Rubenstein 1

Each time you encounter a new type of enzyme, write the general reaction catalyzed by that reaction in the space below and then enter specific examples. This will help you organize your thoughts. 1. Hydrolase 2. Hydratase 3. Kinase 4. Dehydrogenase 5. Dehydratase 6. Aldolase 7. Enolase (Enol hydratase) 8. Mutase 9. Isomerase 10. Oxidase


11. Oxygenase 12. Carboxylase 13. Decarboxylase 14. Aminotransferase 15. Phosphatase

16. Phosphorylase

17. Lyase

III. Important points to focus on when learning a pathway:

A. What is the relation of the overall pathway to other pathways with which it may interact?

B. What is the main function of the pathway in the tissue in which it is being studied? For example, how do the roles of glucose metabolism differ in liver and muscle?

C. What are the biologically irreversible steps in the pathway, are they under

metabolic control, and if so, how? Does the control you think is exerted make sense relative to the function of the pathway?


D. What are the oxidation or reduction steps in the pathway?

E. Which intermediates in the pathway serve as branch points with other pathways?

F. Which steps in the pathway use coenzymes and which ones are used.?

G. In what tissues are the pathways active and in which subcompartments of the cell are these reactions carried out?

H. What are the structures of the intermediates in the pathway, and what are the names of the enzymes that catalyze the reactions?


GLYCOLYSIS AND GLUCONEOGENESIS

Clinical Vignette (NEJM 344, 1588-1592 (2001))

A baby girl of Norwegian ancestry was delivered by cesarean section at 36

weeks gestation (birth weight, 1670 g; length, 42 cm) because of poor fetal growth

(Subject III-7 in pedigree). Her parents were first cousins, and both had glucose

intolerance. In addition to being small for gestational age (< 3rd percentile), the infant

had total situs inversus. On the first day of life, her blood glucose concentration was

145 mg/dl, and on day 2 it was 300 mg/dl at which time treatment with insulin was

started. The initial insulin requirement was 0.75 U/kg body weight per day. Blood

glucose control was difficult to achieve, and there were large variations in blood glucose

concentration (35-630 mg/dl) but no ketosis. Tests for antibodies against insulin,

glutamic acid decarboxylase and protein tyrosine phosphatase like molecule IA-2 (a

major target antigen of cytoplasmic islet-cell antibodies) were negative. Basal and

glucagon-stimulated serum C-peptide concentrations were nearly undetectable on

several occasions. Plasma glucagon concentrations were within the normal range. The

girl had no digestive problems.

When the girl was five years old, epilepsy developed, probably as a sequela of a

neonatal brain abscess, and she subsequently had mild learning and behavioral

difficulties. Her motor development was normal. At the age of 15 years, the glycemic

response to glucagon was normal. Her sister (Subject III-6) presented with typical type

1 diabetes at the age of seven years. Her mother (Subject II-7) was given a diagnosis

of gestational diabetes at the age of 25 yrs. Her father (Subject II-6) had impaired

fasting glycemia that was treated with diet.


Study Guide for Case 1. What is the significance of offspring from first cousins? 2. What is glucose intolerance and what might its causes be? 3. What is situs inversus? Consequences? 4. What are normal blood glucose levels? 5. Why was insulin administered, and why was there no ketosis if blood glucose

remained elevated? 6. Why were tests done for anti-insulin, anti glutamate decarboxylase and anti “II-A”

antibodies? What is the significance of the negative result coupled with the need for insulin?

7. What is C-peptide and why should glucagon stimulate its appearance in the serum.

What is the significance of its inability to do so in this patient? 8. What is the significance of normal plasma glucagon levels in this patient? 9. What is the significance of a normal glycemic response to glucagon in this patient?


LEARNING OBJECTIVES 1. Describe the three major reservoirs of energy used in human metabolism. 2. Describe the substrates and ultimate products of anaerobic and aerobic glycolysis. 3. Explain the different roles of glycolysis in muscle, liver, brain, red blood cells, and

adipose tissue. 4. Write the pathway for glycolysis including names of the enzymes and names and

structures of the metabolic intermediates involved. 5. In general terms, describe the function of a kinase, isomerase, mutase,

dehydrogenase, dehydratase, and aldolase. 6. Explain which enzymes in glycolysis catalyze biologically reversible and irreversible

reactions. 7. Explain what is meant by the first committed step in glycolysis. 8. Describe the four enzymes needed for gluconeogenesis to circumvent the

biologically irreversible steps of glycolysis. Describe how biotin works. 9. Describe how glycolysis and gluconeogenesis are coordinately controlled so that

futile cycling is avoided. 10. Explain how fructose-2,6-bisphosphate metabolism is under hormonal control. 11. Describe the differences between Type I and Type II diabetes and the possible

causes for each.


Key words:

Anabolic Catabolic Anaplerotic Glucose transporter (GLUT) Glycolysis Gluconeogenesis Hexokinase Glucokinase Glucose-6-phosphate Fructose-6-phosphate Phosphoglucoseisomerase Fructose-1,6-bisphosphate Phosphofructokinase Dihydroxyacetone phosphate Glyceraldehyde-3-phosphate Aldolase Isozyme Triosephosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase Nicotinamide coenzyme Flavin coenzyme 1,3-Diphosphoglycerate Phosphoglycerate kinase 3-Phosphoglycerate 2-Phosphoglycerate 2,3-Diphosphoglycerate 3-Phosphoglycerate kinase Phosphoglyceromutase Enolase Phosphoenolpyruvate Pyruvate Pyruvate kinase Lactate Lactic acidosis Lactate dehydrogenase Creatine phosphate Creatine phosphokinase Adenylate kinase Hyperglycemia Hypoglycemia Glucose-6-phosphatase Fructose-1,6-bisphosphatase Pyruvate carboxylase

Acetyl CoA Biotin Phosphoenolpyruvate carboxykinase Fructose-2,6-bisphosphate Fructose-6-phosphate-2-kinase Fructose-2,6-bisphosphatase Glucagon Insulin Hypoglycemia G-protein Adenylate cyclase cAMP Protein kinase A Phosphoprotein phosphatase Diabetes mellitus Hyperinsulinemia Hemolytic anemia Myopathy IGF-1


I. Overview of metabolism

A. Three basic kinds of pathways (groups of reactions that function together to produce a particular end)

1. Catabolic

a. Energy-yielding - used to generate ATP and reduced electron carriers to power other reactions.

b. Oxidative c. Degradative

2. Anabolic

a. Energy-requiring b. Reductive c. Biosynthetic

3. Anaplerotic. Neither degradative or synthetic. Used to refill pools of intermediates necessary to keep anabolic and catabolic pathways running

II. Carbohydrates as sources of energy

A. Caloric content: 4 cal/gm. B. Three sources of sugars for use by the body

1. Dietary sugars 2. Glycogen stores (muscle for muscle cells and liver for whole body) 3. Gluconeogenesis (resynthesis of glucose from lactate, glycerol, and amino

acids derived from breakdown of body protein). C. Major dietary sugars (NUTRITION):

1. Monosaccharides

a. Glucose (dextrose) b. Fructose (fruit sugar) c. Galactose


2. Disaccharides

a. Sucrose (table sugar) - fructose + glucose b. Lactose (milk sugar) - galactose + glucose

3. Complex carbohydrates

a. Starch and b. glycogen (α-1,4 polymers of glucose with α-1,6 branches)

III. Digestion of dietary sugars

A. Monosaccharides go to intestine where taken up by intestinal epithelial cells (Sec. IV)

B. Disaccharides go to intestine where they are broken down by disaccharidases

such as lactase (specific for lactose), maltase and isomaltase (specific for these two sugars respectively. Disaccharidases are hydrolases that split glycosidic bonds joining two sugars.

C. Complex sugars (glycogen and starch)

1. Digestion starts in the mouth

a. Salivary glands secrete amylase, an α-1,4 endoglucosidase

b. Produces oligomers of α-1,4 glucose and branched oligomers containing

α-1,6 branch points.


2. Digestion finishes in the intestine

a. Acid environment of stomach inactivates the enzyme and the products pass into the duodenum

b. Pancreas secretes sodium bicarbonate into the duodenum to neutralize

stomach acid and it also secretes amylase which finishes breaking down the complex carbohydrates to mostly the disaccharides maltose (glucose-α1,4-glucose) and isomaltose (glucose-α1,6-glucose)

c. In jejunum (middle part of small intestine) disaccharidases now finish

digestion to monosaccharides.

i. Disaccharidases are secreted by cells of the jejunum and bind to the lining of the intestine. Lactase splits lactose

ii. Loss of intestinal lining because of injury or diseases (GI infections

leading to diarrhea) can cause deficiencies in these enzymes until the intestinal lining regenerates.

D. Pathology related to carbohydrate digestion

1. Lactose intolerance

a. Intestinal injury or genetic deficiency (people from Asian or American

Indian heritage)

b. Bloating, diarrhea, failure to thrive over long periods of time. c. Control by withholding lactose from diet (difficult) or ingesting lactase pills

(Lactaid).

2. Elevated serum amylase

a. Amylase levels in the blood are normally low to nonexistent. b. Pancreatic disease such as pancreatitis or pancreatic cancer causes

leakiness or disruption in pancreatic cells. c. Result is elevation in serum amylase activity. Higher the enzyme level,

the worse the injury or disease.


IV. Transport of glucose into the cell

A. In mammals, glucose entry occurs via facilitated diffusion through one of a series of glucose transporters in the cell membrane each encoded by a different gene.

B. Four different types of transporters with which you need to be concerned, depending on physiological role of a particular cell:

1. Most cells –GLUT1 and GLUT3 gene products: - non-hormone dependent,

high affinity for glucose.

2. Intestinal uptake of glucose – combined effect of GLUT2 gene product and Na+ - glucose transporter

a. Glut 2 gene product - High Km for glucose. Present in liver, β-cells of the

pancreas, kidney as well as intestinal epithelium. Only allows significant glucose entry or exit in times of high glucose. Facilitates diffusion of glucose from intestinal cell to blood along its concentration gradient

b. Na+ - glucose transporter: transports glucose from lumen of intestine into intestinal cells.

i. Co-transport of Na+ is required ii…Energy for transport of glucose into the intestinal cell against a glucose gradient is provided by entry of Na+ along its electrochemical gradient. iii. Na/K ATPase “pump” maintains inward Na+ gradient.


3. Liver and kidney - GLUT2 - Liver (primarily) and kidney (secondarily) are responsible for glucose homeostasis.

a. Liver makes glycogen in times of high blood glucose and breaks it down

and returns glucose to blood when blood glucose levels drop. b. When blood glucose is low, liver and kidney also manufacture glucose

from amino acid breakdown products and release glucose into blood (gluconeogenesis).

c. Only want glucose taken from blood for storage as glycogen when blood

glucose is high. High Km of GLUT2 receptor allows this limitation

d. Mutation causing GLUT2 deficiency leads to hepato-renal glycogenosis (glycogen accumulation) – condition called Fanconi-Bickel Syndrome. Why?

4. Control of glucose homeostasis (60-90 mg./dl. in serum)

a. Glucagon – signal for low blood glucose (α-cells of pancreas) b. Insulin – signal for high blood glucose (β-cells of pancreas)

5. β-Cells of pancreas: GLUT2 important for insulin secretion.

a. Insulin secretion depends on glycolysis in β-cell b. Rate of glycolysis depends on rate of entry of glucose into cell. c. High Km of GLUT2 receptor means that there will be significant entry of

glucose only during high blood glucose concentrations, exactly the time one would like insulin secreted.


6. Muscle and Adipose tissue - GLUT4 gene product:

a. Activity is stimulated by insulin (promotes fat and protein synthesis).

b. Transporter is in submembraneous compartment. c. Insulin recruits receptors to the cell membrane where they can

facilitate entry of glucose into these cells.

d. Alternative EXERCISE-DEPENDENT INSULIN-INDEPENDENT SIGNAL TRANSDUCTION PATHWAY for recruiting GLUT4 transporters to muscle cell membrane.

i. Increases glucose availability during exercise

ii. the reason why exercise can help lower blood sugar in Type

II diabetics.

e. INSULIN RESISTANCE

i. Decreased glucose clearance from the blood in response to insulin

ii. Associated with Type 2 diabetes and metabolic syndrome

iii. Lipid-induced inhibition of GLUT4 receptor to muscle cell

membrane

a) Elevated palmitate, a C16 saturated fatty acid (Tuschiya, Y. et al. Palmitate-induced down-regulation

of sortilin and impaired GLUT4 trafficking in C2C12 myotubes. 2010. J. Biol. Chem., in press.)

b) Accumulation of acyl-carnitines due to depressed

oxidation of fatty acids. (Noland, R.C., et al. Carnitine insufficiency caused by

aging and overnutrition compromises nitochondrial performance and metabolic control. 2009. J. Biol. Chem. 284, 22840.)


15

General flow chart for central metabolism


V. GLYCOLYSIS - Overall Metabolic Considerations:

A. Oxidation of glucose produces NADH (electrons), ATP, and pyruvate B. Products handled in two ways depending on metabolic state of the cell

1. Aerobic utilization:

a. Electrons transferred to mitochondria b. Pyruvate converted to Acetyl CoA in mitochondria

2. Anaerobic utilization

a. Electrons transferred to pyruvate to make lactate in cytosol. in mammals. Ethanol is made in yeast and other organic acids are made by other microorganisms.

b. Pyruvate + electrons used to make ethanol in cytosol (yeast).


GLYCOLYTIC PATHWAY


VI. Overall Pathway:

A. Priming stage (activation and trapping by phosphorylation)

B. Splitting the sugar into two 3-C fragments and sugar oxidation.

C. Energy extraction (oxidation and ATP formation) Step 1 Phosphorylation of 6 position of glucose (Hexokinase/glucokinase)

A. Biologically irreversible

B. Hexokinase and glucokinase have distinct physiological roles.

1. Glucokinase is about 90% of total glucose phosphorylating activity in the liver. 2. Hexokinase Km for glucose: 50 μM; Glucokinase Km for glucose: 7-10 mM.

Vmax (Hexokinase)<Vm(Glucokinase). 3. Hexokinase is feedback inhibited by physiological concentrations of glucose-

6-P. Glucokinase is not feedback inhibited. 4. Consequences:

a. In nonhepatic tissues, cells must be able to remove glucose from blood and utilize it at all times. Hence, need hexokinase with the low Km so it can work maximally at lower glucose concentrations (Fasting blood levels: 3-5 mM). Since it is essentially always capable of working at full capacity, it must be controlled so that it is not allowed to make more glucose-6-P than the cell can use at a particular time.

a. Since liver regulates blood sugar levels, it needs to be able to remove

glucose when blood glucose levels are high (glycogen and fatty acid synthesis) and return glucose to blood when glucose levels are low (glycogenolysis and gluconeogenesis). Because of the high Km, glucokinase only works efficiently after eating when blood sugar levels are elevated. After fasting, the blood levels drop to the point that they are way


below Km for glucokinase rendering it much less active and permitting net flow of glucose out of the liver cell.

b. Recently shown that in a human lacking glucokinase activity,

administration of glucagon produces a normal glycemic response – liver must have glycogen. Paradox (Defect in our case)? Maybe in this situation, expression of another enzyme is induced. It has been demonstrated that in person with cirrhosis of the liver where glucokinase activity is low, there is a compensatory stimulation of hexokinase 2 activity (Lowes et al in Biochem. Biophys. Res. Comm. 1379, 134 (1998). Maybe this happens here?

c. Glucokinase in the pancreas in β-cells. Essential for proper insulin

secretion. Only isoform present. If insulin secretion depends on glycolysis and insulin should only be made in elevated amounts in the presence of high blood glucose, the best thing to do would be to have glycolysis that works only when blood glucose levels are high. Glucokinase, with its high Km for glucose, fits this role perfectly, and together with the GLUT2 receptor, allows insulin secretion to be tightly coupled to blood glucose levels.

Step 2: Conversion of glucose-6-P to fructose-6-P (phosphoglucose isomerase)

A. Biologically reversible

B. Sets up molecular for bond cleavage between carbons 3 and 4

C. Fructose-6-P is one of two substances that combine to make 5-carbon sugars (Pentose phosphate Pathway).

Step 3: Phosphorylation of F-6-P to make F-1,6-bisphosphate (Phosphofructokinase)


A. Biologically _______________________

B. The committed step in glycolysis and the major point of metabolic control.

1. Strongly inhibited by the binding of ATP to an allosteric site at physiological ATP concentrations. There must be a mechanism for relieving this inhibition, especially in a tissue like liver where (ATP) does not fluctuate much or glycolysis would not work.

2. Activated by 5’-AMP (non-covalent) 3. Inhibited by acidic pH values (Consequences for heart attack?)

C. Pathway is energy yielding but so far used up two ATPs.

D. End of priming stage Step 4: Splitting of F-1,6-P2 into Dihydroxyacetone-P (DHAP) and Glyceraldehyde-3-

P (Aldolase)

A. ΔGo >> 0, but ΔG is about 0 - Biochemical push- pull


B. There are three isozymes of fructose-1,6-P2 aldolase: A, B, and C

1. A: Muscle 2. B: Liver (also works on fructose-1-P; the only isozyme that does 3. C: Brain 4. Advantages of isozymes: Body can have a defective gene expressed in one

tissue or cell without affecting the function of a particular cell or the other tissues in the organism.

C. At this point, nature

Converts one 3-C fragment to the other allowing the entire pool to be converted to pyruvate via one pathway.

D. DHAP is a precursor for glycerol-P synthesis, necessary for triacylglyceride (storage fat) and membrane P-lipid synthesis.

E. Glyceraldehyde-3-P is a precursor for the synthesis of 5-carbon sugars (Pentose-P Pathway) along with fructose-6-P.

F. Glyceraldehyde-3-P is the entry point for fructose carbons into mainstream

metabolism. Step 5: Interconversion of DHAP and Glyceraldehyde-3-P (Triose-P isomerase)

A. Reversible B. What happens to ATP yield in aerobic and anaerobic glycolysis if the enzyme is deficient?


B. The 1-carbon oxidation reaction per se is highly exothermic. How can you tell this?

C. Wherever there is an oxidation or reduction rxn, there is usually a regeneratable electron acceptor or donor (usually a nicotinamide coenzyme-NAD(H) or NADP(H)- or flavin coenzyme- FAD(H2) or FMN(H2).

1. NAD and NADP come from the vitamin niacin. Deficiency causes pellagra 2. FAD and FMN come from the vitamin riboflavin (vitamin B2)

D. Rxn. represents the fixation of inorganic phosphate into an organic molecule. Hence, a shortage of Pi inhibits glycolysis.

E. Rxn. is reversible under in vivo conditions.

F. Arsenolysis: When AsO4, a Pi analogue, is substituted for phosphate, it makes arseno anhydride at C1 during this rxn. However, this is very unstable in water and quickly hydrolyzes to produce 3-phosphoglycerate. Thus, energy generated in the oxidation is dissipated meaning that no ATP can be made from 1,3-DPG – example of uncoupler


A. A reversible kinase rxn.

B. Example of substrate level ATP synthesis (not involving the electron transport chain).

C. Two molecules of ATP per mole of glucose produced at this step - two 3-carbon fragments being metabolized.

Step 8: Conversion of 3-P-glycerate to 2-P-glycerate (Phosphoglyceromutase)


4. For every molecule of 2,3-BPG formed, bypass the phosphoglycerate kinase rxn. and lose 1 ATP.

C. Rxn. is reversible Step. 9: Conversion of 2-Phosphoglycerate to Phosphoenolpyruvate-PEP (Enolase)

A. Rxn. is reversible

B. Rxn is inhibited by fluoride

C. Rxn. produces a molecule with a very large negative free energy of hydrolysis (-14 kcal./mole).

Step 10. Conversion of PEP to pyruvate + ATP (Pyruvate kinase)


A. Biologically irreversible

B. Large negative ΔG pulls the entire pathway toward completion. Two kinases

(hexokinase and PFK) at top of the pathway push the pathway thermodynamically.

C. Pyruvate kinase strongly inhibited by ATP and by Alanine, a transamination product of pyruvate. Feedforward activated by fructose-1,6-P2 (activity at top of the pathway activates the bottom part of the pathway).

D. In liver, pyruvate kinase also controlled by covalent modification - phosphorylation by cAMP-dependent protein kinase inhibits the enzyme. Makes it even more sensitive to ATP that it already is.

E. Two ATP produced here per mole of glucose metabolized. Hence, per

glucose thru glycolysis, the path expends two molecules of ATP but makes four for a net production of 2 ATP.

Step 11 Anaerobic regeneration of NAD from NADH - Reduction of pyruvate to lactate

by lactate dehydrogenase (LDH)

A. For every molecule of glucose that goes to pyruvate, 2 molecules of NADH are

generated. B. If NADH is not converted back to NAD, glycolysis will stop C. Reaction is reversible depending on the oxidation state of the cell. In an aerobic

cell, lactate is taken from blood, converted back to pyruvate by oxidation, and the pyruvate can then be subsequently used for energy generation or biosynthesis.


D. Question: In which of the following cell types would a LDH deficiency be the

most devastating? Why? Brain, Skeletal Muscle, Liver, Red Blood Cell.

E. LDH: important serum marker for cell lysis, especially for muscle and liver.

F. Elevated serum lactate levels means in the body, oxidative metabolism is not sufficient to meet normal metabolic demands of the body for energy production. In severe cases, get lactic acidosis – elevated serum lactate plus a drop in serum pH.

CARBOHYDRATECARBOHYDRATE--DERIVED ENERGY DERIVED ENERGY

GlycogenGlycogen

GG--11--PP GG--66--PP GlycolysisGlycolysis 2 2 PyrPyr

MitoMitoADP + PADP + Pii ATPATP

2 NAD2 NAD 2 NADH2 NADH

LactateLactate

(Aerob)

(Anaerob)

Blood Blood GlucoseGlucose

cAMPCa+2

5’-AMP

VII. Tissue specific uses and fates of glucose

A. Brain: aerobic glycolysis acetyl CoA etc. - Under normal conditions, glucose is virtually the only carbon source used. Ketone bodies from fatty acids can be equally well used after 3-4 days of starvation.

A. Muscle: aerobic or anaerobic depending on the exercise state of the muscle and its oxidative capacity. The redder the muscle, the more mitochondria it has, and the more it can rely on oxidative metabolism which is more energy efficient.

B. Red Blood Cell: totally anaerobic glycolysis. No mitochondria in the cell so it

can’t do Krebs Cycle and Ox. Phos. Lactate is produced and dumped into the blood where it is taken up by non-hepatic tissues and further oxidized or taken up by the liver and converted back to glucose (gluconeogenesis).

D. Liver: aerobic, largely. Derives its energy from oxidation of fatty acids. Uses excess glucose via glycolysis to make fatty acids for subsequent storage of fat in adipose tissue. During conditions of low blood glucose, glycolysis is turned off


and gluconeogenesis is used to make glucose for export into the blood. Many of the same enzymes are used in both directions. Also provides a means for replenishing intermediates of the Krebs Cycle using sugar carbons.

E. Adipose tissue: Glucose is split into three carbon fragments which are

converted to a derivative of glycerol to provide the backbone for triacylglyceride (storage fat) synthesis.

VII. Energy metabolism in the heart and skeletal muscle

A. Resting and moderately exercised skeletal muscle and virtually all cardiac muscle: oxidation of fatty acids to make ATP (mitochondria).

B. If oxidative capacity of muscle exceeded, two ways remain to derive energy: readily available reservoir or glucose from glycogen breakdown.

C. Readily available metabolic energy (ATP) in muscle:

1. ATP per se - 20% (ΔGo hyd. = -8 kcal/mole) NH2

+ 2. Creatine phosphate - 80% (ΔGo hyd = -10 kcal/mole)

P NH C N CH2 CO2-

a. Made by creatine phosphokinase (CPK) CH3

Creatine-P + ADP Creatine + ATP

b. When energy supply is ample, rxn. is driven toward creatine-P synthesis. When energy production slows, creatine-P is used to make ATP.

3. Adenylate kinase (myokinase)

2 ADP AMP + ATP

B. Some glycogen in cardiac muscle, a lot in skeletal muscle. Converted to glucose-6-P for use in glycolysis during time of energy need.


C. Under normal conditions, heart, and to a lesser extent skeletal muscle produce energy oxidatively to the extent it is possible since oxidative metabolism (using fatty acids, ketone bodies, lactate, and glucose as fuels) produce more ATP per molecule of fuel metabolized than does anaerobic glycolysis.

D. Oxygen deprivation to the heart rapidly exhausts reservoirs of energy in the heart

since to chance for oxidative metabolism. Heart must then revert to anaerobic glycolysis. Lactate accumulates (lactic acidosis)-(pain) slowing down glycolysis (PFK) making the situation worse metabolically. Also, for major heart attacks, since heart is not pumping efficiently, it is harder for the body to eliminate the lactate from the cardiac muscle.

___________________________________________________________________ VIII. GLUCONEOGENESIS - Resynthesis of glucose from lactate (liver and kidney)

or from amino acid breakdown products or from glycerol.

A. Two reasons to rely on gluconeogenesis.

1. In times of hypoglycemia and lowered glycogen stores, the body tears down muscle protein first and then serum proteins to generate precursors for glucose production. 2. Red blood cells and other tissues doing anaerobic glycolysis deliver lactate to the blood. Much of it enters the liver where it is reconverted to glucose and released back into the bloodstream.

B. If the process is largely a reversal of glycolysis, which enzymes in glycolysis cannot be used for this pathway? C. Metabolic detours used instead

1. Glucose-6-phosphatase

a. A multi-subunit complex that works inside the ER.

b. A deficiency in any subunit can cause a deficiency in the activity.


2. Fructose-1,6-bisphosphatase (works opposite PFK1)

Coordinately controlled opposite to that of PFK so glycolysis and gluconeognesis do not fully operate at the same time.

3. Pyruvate PEP - Requires energy of two high energy phosphate bonds (2 enzymes needed: pyruvate carboxylase and PEPCK)

a. Pyruvate carboxylase:

i. Requires biotin as a carrier of active CO2 and needs Acetyl CoA as an allosteric activator. Mitochondrial.

Also allows glucose carbons to be used to generate OAA, a carrier for acetyl CoA in the Krebs Cycle.

ii. Biotin is covalently bound to a number of different carboxylases and participates as an active carboxyl donor in carboxylation reactions:


b. PEPCK (PEP-Carboxykinase):

i. Two isoforms: cytosolic and mitochondria. Cytosolic form drives gluconeogenesis. – Function of mitochondrial form not well understood. (For newest research: Stark, R., Pasquel, F., Turcu, A., et.al. (2009) J. Biol. Chem., in press)

c. For amino acid breakdown products leading through Krebs Cycle to oxaloacetate, pyruvate carboxylase is not necessary for gluconeogenesis. PEPCK is always needed whether you are starting with a 3-C (at the level of pyruvate) or 4-C substrate (Krebs Cycle intermediate).

D. Gluconeogenesis is an energy-requiring pathway

a. pyruvate OAA: 1 x 2 = 2 ~Ps b. OAA PEP: 1 x 2 = 2 ~Ps c. 3-P-glycerate + ATP 1,3-Bisphosphoglycerate + ADP: 1 x 2 = 2 d. 1,3-Bisphosphoglycerate + NADH Glyceraldehyde-3-P + NAD. e. Per mole of glucose synthesized, one needs two molecules of 3-C and/or 4-

carbon substrates. Each amino acid molecule contributes at most 1 of these fragments.


IX. Diabetes mellitus (most common defect in regulation of carbohydrate metabolism)

A. Manifestations

1. Elevated blood glucose levels – water drawn from interstitial space to reduce osmotic imbalance caused by high blood glucose. Leads to:

2. Loss of glucose in urine with excessive urine and thirst

3. Fatigue, nausea, vomiting (acute onset)

4. Long-term uncontrolled disease leads to blindness, neuropathy, and vascular disease.

5. Accelerated release of and oxidation of fatty acids sometimes leading to ketoacidosis (generally Type 1).

6. Decreased insulin leads to increased glucagon which exacerbates

hyperglycemia (stimulates gluconeogenesis and subsequent body protein wasting) .

B. Type I diabetes

1. Insulin-dependent due to loss of insulin secreting β-cells of the pancreas or deficiency in one of the enzymes controlling insulin secretion.

a. Neonatal – enzyme deficiency.

b. Juvenile or sometimes adult onset- autoimmune in origin causing β-cell

destruction with a frequency of 1/200.

2. Hyperglycemia and other symptoms are due to inablility of muscle and adipose tissue to utilize blood glucose because of suppressed glucose transport. Short term acute metabolic problems and long term problems due to destruction of blood vessel endothelium

C. Type II diabetes

1. Used to be called adult onset- often associated with obesity. Now a large

number of teenagers and young adults are presenting with this disease. 2. Insulin independent initially. In later stages can become insulin dependent-

insulin is made but is not biologically effective generally because of densitized insulin receptor system.


3. Insulin resistance causes additional insulin to be release (hyperinsulinemia) which then activates IGF-1 receptors (lower affinity for these than insulin receptors but lowered affinity overcome by increased amount.). Promotes proliferation of smooth muscle cells in vascular wall aiding in build-up of occlusive deposits.

d. Incidence about 5/100 - Control with diet and drugs.

X. CONTROL OF GLYCOLYSIS AND GLUCONEOGENESIS.

1. Hexokinase and glucokinase (already discussed above)

2. Phosphofructokinase (Liver) and Fructose-1,6-Bisphosphatase

a. PFK1-Catalyzes the committed step in glycolysis b. PFK1 - Extremely sensitive to inhibition by normal levels of ATP. Inhibition

must be relieved if enzyme is to be active. c. Allosteric site for Fructose-2,6-Bisphosphate on PFK1 - Prevents binding of

ATP at the control site of PFK1 leading to activation of PFK) so that its Km for F-6-P is decreased. (Kd of F-2,6-P2 for PFK1 is about 10 nM. F-2,6-P2 also binds to fructose-1,6-bisphosphatase and inactivates it.

d. Levels of F-2,6-P2 are hormonally controlled by glucagon: F-2,6-P2 are high

during high blood sugar and low during low blood sugar.

i. Fructose-6-P-2-kinase makes F-2,6-P2

ii. Fructose-2,6-bisphosphatase destroys F-2,6-P2


iii. Fructose-2,6-bisphosphate synthesis and degradation are catalyzed by different active sites on the same polypeptide chain. Relative activity controlled by covalent phosphorylation by protein kinase (kinase A).

♦ Unphosphorylated protein: 2-kinase is active and 2,6-bis-

phosphatase inactive - Glycolysis is favored

♦ Phosphorylated protein: PFK2 is inactive and 2,6-bisphosphatase is active - Gluconeogenesis is favored

Fructose-2,6-bisphosphate control system (Liver) Low Blood Sugar Glucagon Glucagon Glucagon Glucagon Receptor Receptor Aden. Cyc. Gα GTP GDP #Gα Gβγ ATP #Gα- Aden. Cyclase R Kinase A cAMP ------#K----P R- cAMP Fruc.-6-P P-Prot. Kinase A# Fruc.-2,6-P2 Phosphat. P ------K----#P Glycolysis Glycolysis Gluconeo Gluconeo


When unphosphorylated, 2-kinase is active and 2,6-bisphosphatase is inactive: F-2,6-P2 increases.

• When phosphorylated, 2-kinase is inactive and phosphatase is active: F-2,6-P2 decreases.

iv. Phosphorylation of the bifunctional enzyme is carried out by cAMP-

dependent protein kinase (kinase A).

• Glucagon binds to receptor on the cell membrane

• Receptor-ligand complex activates a trimeric GTP-binding protein causing the α-subunit to exchange GTP for GDP (inactive).

• Activated α-subunit binds to and activates adenylate cyclase (cell membrane) which makes cAMP.

• cAMP binds to inactive kinase A releasing the catalytic subunit which

now phosphorylates its target proteins on selected serine and threonine residues.

e. In cardiac and skeletal muscle, fructose-2,6-bisphosphate is always present

at high enough levels so that ATP does not shut down glycolysis. The bifunctional enzyme in skeletal muscle is not negatively regulated by cAMP (synthesis of which is stimulated by adrenalin, a stress hormone) so that the increased energy needed in times of stress can be provided by glycolysis. Increased ADP levels following muscle contraction, then allows glycolysis.

3. Pyruvate kinase regulation(discussed in part above)

a. Liver enzyme is inhibited by covalent phosphorylation - makes the protein

even more sensitive to ATP.

b. Phosphorylation is carried out by A-kinase in response to elevated cAMP induced by the glucagon released into the bloodstream during hypoglycemia.

c. During normoglycemic conditions, glucagon release is terminated, and constitutive phosphoprotein phosphatases in the cell remove the phosphates from pyruvate kinase allowing glycolysis to occur once more.


MEDICAL AND PHYSIOLOGICAL ISSUES IN CHAPTER NOT TO BE NEGLECTED 1. Fanconi-Bickel Syndrome (GLUT2 receptor deficiency) 1. Insulin-dependent glucose uptake (GLUT4) 2. Neonatal diabetes (glucokinase deficiency) 3. GLUT2, Glucokinase and insulin secretion 4. Gluconeogenesis and glucose homeostasis 5. Anaerobic metabolism and lactic acidosis 6. Muscle energy metabolism (available energy stores) 8. Roles of the pancreatic hormones insulin and glucagons 9. Type 1 and Type 2 diabetes 10. Insulin resistance


SUMMARY FOR GLYCOLYSIS AND GLUCONEOGENESIS

(I)

1. Glucose enters the cell via facilitated diffusion mediated by products of the GLUT genes. All are constitutively active except for the GLUT4 receptor, present in muscle and adipose tissue, whose activity is stimulated by insulin resulting in increased glucose uptake in these tissues.

2. Glycolysis is an energy yielding pathway (ATP) that converts glucose to two moles of pyruvate. Anaerobically, pyruvate is converted to lactate which is released into the blood. Aerobically, pyruvate is converted to acetyl CoA in the mitochondrion and is further oxidized for energy production.

3. Most tissues use glycolysis for energy production (brain, muscle, red blood cell, etc.) Because liver functions to control blood glucose levels, it uses glycolysis in times of high blood glucose to convert excess carbohydrate into fatty acids. The liver uses fatty acid oxidation for energy production.

4. The “priming stage” of glycolysis involves the conversion of glucose to fructose-1,6-bisphosphate via reactions catalyzed by hexokinase (glucokinase), phosphoglucose isomerase, and phosphofructokinase-1 (PFK1). The two kinase steps are biologically irreversible and together consume 2 moles of ATP/mole of glucose processed.

5. Glucokinase is in liver, kidney, and β-cells of the pancreas. Its high Km for glucose (7-10 mM) means it is only effective at high blood sugar levels. Hexokinase has a low Km for glucose (50 μM) and is feedback inhibited by glucose-6-phosphate to prevent accumulation of more of this intermediate than can be processed by the cell.

6. The conversion of fructose-6-P to fructose-1,6-P2 by PFK1 is the first committed step of the pathway, even though it is the second biologically irreversible step, and is the major point at which metabolic regulation of the pathway is exerted. Glucose-6-P, the product of the first kinase, is a substrate for both glycogen synthesis and the pentose-phosphate shunt. By controlling glycolysis at the second kinase, the cell can still synthesize glucose-6-P for these other purposes even when glycolysis is inhibited.


SUMMARY FOR GLYCOLYSIS AND GLUCONEOGENESIS

(II)

1. In the “splitting stage”, aldolase converts fructose-1,6-P2 reversibly to glyceraldehyde-3-P and dihydroxyacetone-P (DHAP). Three isoforms of the protein exist and are expressed in a tissue-specific pattern. Isozymes provide a survival advantage for the cell. The liver isoform, aldolase B, is the only one which can also use fructose-1-P, an intermediate in the metabolism of fructose by the liver.

2. Triose-P isomerase interconverts the two three carbon fragments DHAP and glyceraldehyde-3-P. Glyceraldehyde-3-P can then be used for the subsequent steps in glycolysis.

3. Glyceraldehyde-3-P is an intermediate that joins glycolysis with both the pentose-phosphate shunt and with fructose metabolism. DHAP is reversibly converted to glycerol-P which is needed for membrane lipid synthesis and for synthesis of triacylglycerides (TAGs).

4. The “energy extraction stage” begins with the conversion of glyceraldehyde-3-P to 1-3-bisphosphoglycerate catalyzed by glyceraldehyde-3-P dehydrogenase, the only oxidation step in the pathway. NADH is produced and the large amount of free energy liberated by aldehyde oxidation drives the synthesis of a high energy mixed anhydride compound, 1,3-bisphosphoglycerate, from inorganic phosphate. This mixed anhydride is used as a phosphate donor by 3-phosphoglycerate kinase. Both steps are reversible. 1,3-bisphosphoglycerate is also used as a precursor for 2,3-BPG production, important in the red blood cell for modulation of O2 binding to hemoglobin and in all cells for activation of phosphoglyceromutase.

5. 3-Phosphoglycerate is ultimately converted to phosphoenolpyruvate (PEP) by phosphoglyceromutase and enolase. PEP is a high energy phosphate donor used by pyruvate kinase irreversibly to synthesize ATP and pyruvate. The free energy released pulls glycolysis forward. Per mole of glucose there is a net synthesis of two moles of ATP over the entire pathway.

6. The NADH produced by the glyceraldehyde-3-P dehydrogenase must be reconverted to NAD to allow glycolysis to continue in the cytosol. Anaerobically, the cell uses NADH electrons to reduce pyruvate to lactate via lactate dehydrogenase (reversible). Aerobically, NADH electrons are shuttled into the mitochondrion where they generate more ATP following their passage down the electron transport chain.

7. In muscle, high energy phosphate is stored as creatine-P. Creatine phosphokinase (CPK) reversibly interconverts these compounds depending on the cell’s energy state. Muscle cell death releases CPK into the blood and serum CPK is thus useful for diagnosing heart attacks and muscle injury or disease.


SUMMARY OF GLYCOLYSIS AND GLUCONEOGENESIS (III)

1. Liver, primarily, and the kidney cortex, carry out gluconeogenesis, the resynthesis of glucose from pyruvate and/or oxaloacetate in order to raise blood glucose levels or to decrease serum lactate or alanine levels (both are converted to pyruvate). Oxaloacetate is derived from the breakdown of amino acid carbon skeletons following degradation of muscle proteins.

2 Generally, the pathway follows the reverse of glycolysis except that the three biologically irreversible steps must be bypassed. PFK is bypassed by fructose-1,6-bisphosphatase, glucokinase is bypassed by glucose-6-phosphatase, an ER protein complex which produces free glucose that can then transverse the organ plasma membrane and enter the blood stream. The pyruvate kinase step is bypassed by a combination of two enzymes: pyruvate carboxylase in the mitochondrion which requires biotin as a cofactor and makes oxaloacetate and PEP carboxykinase which utilizes GTP to convert OAA to PEP.

3. There must be an input of both ATP and NADH to reverse the phosphoglycerate kinase/glyceraldehyde-3-phosphate dehydrogenase steps.

4. A total of 6 high energy phosphate bonds are needed to convert two pyruvate to glucose whereas only 4 are needed to convert two oxaloacetates to glucose.

5. The coordinate control of glycolysis and gluconeogenesis in liver depends on the level of fructose-2,6-bisphosphate which stimulates PFK and inhibits fructose-1,6-bisphosphatase. Levels of this intermediate are controlled by a bifunctional enzyme containing both a fructose-6-P 2-kinase and a fructose-2,6-bisphosphatase active site. When the polypeptide is phosphorylated by cAMP-dependent protein kinase following glucagon elevation in the blood as a result of low blood glucose, fructose-2,6-bisphosphate levels drop, glycolysis is turned off, and gluconeogenesis is turned on.

6. In cardiac muscle, a different bifunctional protein gene is expressed. This produces a protein whose 2-kinase activity is stimulated, not inhibited, by elevated cAMP which is produced in the heart in response to elevated adrenalin in the blood.

7. In type I diabetes, the pancreas ceases to produce insulin so sugar uptake by muscle and adipose tissue is inhibited. The increased glucagon that results from lack of insulin increases gluconeogenesis leading to a further elevation in blood glucose which produces long-term pathological effects if not corrected.


RECENT GLUCONEOGENESIS REFERENCES

1. Poelje, PD, Dang, Q, and Erion, MD (2007) Discovery of fructose-1,6-

bisphosphatase inhibitors for the treatment of type 2 diabetes. Curr. Opin. Drug Discov. Devel. 10, 430-437.

2. Rodgers, JT, and Puigserver, P. (2007). Fasting-dependent glucose

and lipid metabolic response through hepatic sirtuin 1. Proc. Nat. Acad. Sci., USA 104, 12861-12866.

3. Sekine, K., Chen, Y.R., Kojima, N., Ogata, K., Fukamizu, A., and

Miyajima, A. (2007) Foxo1 links insulin signaling to C/EBPalpha and regulates gluconeogenesis during liver development. EMBO J. 26, 3607-3615.

4. Dentin, R., Liu, Y., Koo, S.H., Hedrick, S., Vargas, T., Heredia, J.,

Yates, J. 3rd, and Montminy M. (2007) Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2. Nature 449, 366-369.

5. Choi, C.S., et al. (2007) Suppression of diacylglycerol

acyltransferase-2 (DGAT2) but not DGAT1 with antisense oligonuclotides reverses diet-induced hepatic steatosis and insulin resistance. J. Biol. Chem. 282, 22678-22688.

6. Savage, D.B., Petersen, K.F., and Shulman, G.I. (2007) Disordered

lipid metabolism and the pathogenesis of insulin resistance. Physiol. Rev. 87, 507-520.

7. Befroy, D.E., et al. (2007). Impaired mitochondrial substrate

oxidation in muscle of insulin-resistant offspring of type 2 diabetic patients. Diabetes 56, 1376-1381.

8. Petersen, K.F., et al. (2007). The role of skeletal muscle insulin

resistance in the pathogenesis of metabolic syndrome. Proc. Nat. Acad. Sci., USA 104, 12587-12594.


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Dissection of a Metabolic Pathway