BIOCHEMISTRY

13. THE CONJUGATION OF CARBOHYDRATES TO EACH OTHER & PROTEINS

Carbohydrate Structures

Aldehydes vs. Ketones

Stereoisomers

a-maltose Disaccharides a-lactose

Sucrose

1. Produces either an (below) or -glycoside (above) depending on oxygen position a. Determines if digestion occurs (as beta in cellulose cant digest) 2. Polymerization involves activated sugar nucleotide intermediates (e.g. UDPGalactose) 3. Maltose: two glucoses in an (14) linkage 4. Lactose: galactose & glucose in (14) linkage 5. Sucrose: glucose & fructose (-D-glucosyl--D-fructoside)
FAMOUS POLYSACCHARIDES: Saccharide polymers: GLYCOGEN GLYCOGEN THE STORAGE FORM OF GLUCOSE

a forms are when the OH is below the plane at C1; b forms when it is above. Amylose, Amylopectin, Cellulose

Stryer, Biochemistry

6. Glycogen: homopolymers of glucose; chains of glucose with (14) linkages with closely spaced branching via (16) linkages every 8-10 residues; spherical particles in cytoplasm 7. Starch: homopolymers of glucose a. Amylose: unbranched chain with (14) linkage helical coil b. Amylopectin: (14) linkages but is branches every 12 residues via an (16) linkage 8. Cellulose: Unbranched chain with (14) linkages 9. Glycosaminoglycans (GAGs): long unbranched polysaccharides of repeating anionic disaccharides that may be sulfated; important part of connective tissue a. Negatively charged (highly hydrated) b. E.g. Chondroitin sulfate; keratin sulfate; heparin; hyaluronic acid

10. Glycation: proteins reacting with sugars; non-enzymatic involving Schiff base formation between the aldehyde of the sugar and an amino group on the protein to generate a ketoamine; no cofactors
a. b. Is for secreted proteins or domains facing extracellularly (except for O-GlcNac) Can be N-linked (Asn) or O-linked (Ser, Thr)

c. E.g. diabetic Glycated Hemoglobin (HbA1C) gives an integrated average of blood sugar level over the past two months 11. Glycoproteins: critical for biological recognition (e.g. blood types; influenza virus) a. Exported out of the cell or have their glycosylated domains oriented outside (except for O-GlcNac) b. O-GlcNac glycoproteins are found in the nucleus and cytoplasm; regulate cellular responses to hormones as well as regulate transcription, cell growth & division
12. O-linked glycoproteins have carbohydrate added to the protein one sugar at a time a. Are glycosylated in the Golgi b. E.g. blood group substances; salivary musins 13. N-linked glycoproteins have carbohydrate first added as a preassembled 14 sugar block a. First stage is formation of a branched oligosaccharide on a lipid carrier molecule (dolichol phosphate) b. Dolichol is phosphorylated by a kinase c. Synthesis is begun by the sequential transfer of a residue from UDP-N-acetylglucosamine to the phosphate of dolichol-phosphate on the ER membrane

d. Tunicamycin: antibiotic that selectively inhibits the addition of Nacetylglucosamine-phosphate to dolichol phosphate
14. Blood groups are dependent on sugar residues at the ends of carbohydrate chains of glycoproteins or glycolipids

a.

Type O individuals are more prone to ulcers because H.Pylori binds specific structures that look like type O sugars

15. Proteoglycans: consists of negatively-charged polysaccharide glycosaminoglycan chains which are attached to a core of protein a. E.g. Heparin sulfate, collagen, and laminin are interwoven in the basement membrane 16. Selectin- Carbohydrate system a. Recruitment of WBC into areas of infection b. Leukocyte-adhesion deficiency II i. Many infections early in life; inability to create pus at wound site; dont recruit WBCs ii. Inability to put fucose onto things (to make something the selectins will recognize) iii. Abnormal blood type (Bombay) 17. Congenital diseases of glycosylation (CDGs): patients are defective in one or more enzymes that break down glycoproteins; different clinical presentations
a. b. Lysosomes have both endo and exoglycosidases Enzyme deficiencies result in accumulation of partially degraded materials in vesicles that impair function; partial breakdown products are excreted in urine

c. Single cell defects i. Pompe:

1. 2. Glycogen storage disease type II Accumulation of glycogen in the lysosome due to deficiency of the lysosomal acid alpha-glucosidase enzyme. It is the only glycogen storage disease with a defect in lysosomal metabolism

d. Multiple enzyme defects i. I Cell: Failure of the M6P system leads to export of lysosomal enzymes
1. Normally, N-linked glycoproteins can be modified to generate mannose 6phosphate, which is recognized by a receptor in the endosomal trafficking system that causes the modified protein to be transported to the lysosome

14. DIGESTION, ABSORPTION, AND CELL UPTAKE OF CARBOHYDRATES

The Uses/Trafficking of Glucose
UDP-GLUCURONIC ACID
Glycogen Metab

GLUCURONIDES Bilirubin, drugs

GLYCOGEN

UDP-GLUCOSE Glycoprot, Polysacch., GLUCOSE-1-P Gal Metab.

PP Shunt

GLUCOSE

GLUCOSE-6-P
Gluconeogenesis

PENTOSE-5-P
Glycolysis

PYRUVATE CO2, H2O

TCA Cycle

1. Monosaccharides: glucose, fructose

2. Disaccharides a. Maltose (Glu-Glu) b. Lactose (Gal-Glu) c. Sucrose (Fru-Glu) 3. Polysaccharides a. Starch (Glu) i. Amylose is the linear polyglucose ii. Amylopectin is the branched polyglucose 4. Carbohydrate digestion starts in the mouth and continues in the intestine a. Salivary Amylase is inactivated by low pH of stomach (stomach doesnt
digest carbohydrates) i. The end products of starch digestion by Amylase are: 1. Maltose, maltotriose, and small branched oligosaccharides (limit dextrins) a. Are cleaved by specific enzymes present in the brush border membranes of mucosal cells of the small intestine 2. NOT glucose
a-AMYLASE

Name -Amylase -Amylase Maltase Isomaltose

Origin Salivary glands Pancreas Intestinal mucosa Intestinal mucosa

Sucrase Lactase

Intestinal mucosa Intestinal mucosa

Acts on Glu-(14)-glu in starch Glu-(14)-glu in starch Glu-(14)-glu in maltose Glu-(16)-glu in isomaltose and branched dextrins Glu-(12) -fru in sucrose Gal- (14)glu in lactose

a-AMYLASE

SUCRASE LACTASE ISOMALTASE MALTASE

5. Lactose intolerance: Failure to digest lactose; lactase insufficiency a. Can be diagnosed by failure to observe glucose increase after lactose challenge; can observe H2 in the breath
b. Normally: Lumen of intestine: lactose glucose and galactose

c. Intolerance: lactose passes further down GI tract and its metabolism by bacteria in colon formation of gas i. These compounds have an osmotic effect of sucking water out from intestinal cells 6. Mammals lack enzymes to digest cellulose (has glu-(14)glu bonds) and many other plant polysaccharides of dietary fiber
a. Mammalian enzymes that cleave starch and glycosidic bonds of galactose, mannose, pentoses, or sugar acids (from pectin, gum, agar)

b. E.g. xylose; arabinose 7. Facilitated diffusion

a. Mediated by specific membrane carrier proteins and is saturable (the number of carrier protein molecules limits the rate transport as solute concentration increases)
b. c. Does not require metabolic energy Requires a concentration gradient maintained by rapid removal of the transported sugar from inside the cell to the capillaries E.g. fructose; mannose; also glucose absorption into intestinal cells (in addition to active transport in intestine) glucose release is facilitated transport, first stop is the liver

d.

8. Active transport a. For glucose & galactose in intestine b. Allows absorption against a concentration gradient c. The entry of sugar is coupled to the entry of Na+ (for which there is always a gradient of concentration from the outside [high] to the inside) d. Sugar & Na+ are carried by the same carrier protein e. At a different site on the cell membrane, energy is expended (via hydrolysis of ATP) by the cell to maintain the Na+ concentration gradient (to pump Na+ out) i. Membrane moves K+ into the cell while moving Na+ out
SIMPLE DIFFUSION, FACILITATED DIFFUSION AND ACTIVE TRANSPORT.
Basolateral Serosal Carrier facilitated transport

Monosaccharides are transported by:

LUMEN Glucose
Cotransporter Metabolism in epithelial cell Diffusion

Na+
SGLT (Sodium-linked) Intestinal mucosa Kidney brush border

Facilitative transporter GLUT4, an important in fat and muscle (NOT LIVER) cells is regulated by insulin; GLUT2 is not.

[glucose] Transport

Lumen Medium Active Cell

Cell Blood High Medium FacilitatedBlood FacilitatedCell

Other cells Low

9. Transporters a. SGLT (Sodium-linked)/ active transport i. Intestinal mucosa; Kidney brush border b. Facilitative i. Responsive to insulin (GluT4) 1. Skeletal muscle, fat, WBC ii. Not responsive to insulin

1. GluT 2: liver, pancreas, intestine 10. Liver, RBC, and brain cells: carrier-mediated transport is not stimulated by insulin 11. Muscle, fat, and WBC: glucose absorption is stimulated by insulin which acts by mobilizing the transporters from stores on the membranes of the ER a. Insulin reduces circulating THE EFFECT OF INSULIN ON BLOOD GLUCOSE: blood glucose by stimulating Type I Diabetes uptake i. Promotes generation of fat & storage of glucose as glycogen, and decrease of PGL
b. Unstimulated cells have an increased ability to transport glucose in the presence of insulin
The rise in concentration reflects: i. Absorption into the blood ii. Removal from the blood by all tissues iii. Conversion of glucose to lactate, glycogen, and fat in the liver iv. Conversion of non-glucose substrates to glucose in liver v. Return of glucose to blood from liver
Plasma Glucose mg/dL

12.

Diabetics &Blood Glucose Level

a.

b.

Doubling of glucose after a meal; return within 2 hours

Time (min)

c. Diabetes: start higher, go higher, come down slower 13. Lack of an increase in BGL after ingestion of meal with free glucose would indicate a a. Defect in absorption of glucose from intestine 14. Lack of an increase after ingestion of maltose could reflect defect in either a. Digestion of the disaccharide to free glucose b. Or absorption of the glucose
INSULIN ACTIONS IN GLUCOSE METABOLISM
1. decreases blood glucose by increasing uptake in muscle and adipose cells (NOT LIVER); 2. increas es glycolysis in the liver, increasing acetyl CoA formation; 3. decreases gluconeogenic reactions; 4. decreases glycogen breakdown and increases synthesis;

NO INSULIN

+ INSULIN

GFP-GLUT4

15. Glucose is phosphorylated quickly when it is absorbed by isoenzymes (glucokinase or hexokinase) a. G6P is charged and cannot exit the cell Enzymes Hexokinases Distribution All tissues Km for glucose <0.2 mM 10-20 mM Effect Low Km (high affinity; essentially always on) High Km; High Vmax high capacity but low affinity Regulation Feedback inhibition by product (G6P) Substrate concentration and amount of enzyme; Most active when BGL is high

Glucokinase

Liver, pancreatic islet -cells

16. Hexokinase
a. b. c. Can phosphorylate other hexoses (e.g. fructose) High affinity for glucose (km=.1mM vs 5mM glucose concentration) i. Enzymes are always fully on; most tissues always need steady supply of G6P to remain on If cell has enough G6P, it doesnt need more (product inhibition)

17. Glucokinase a. Found in liver (first look at high glucose after a meal; makes even more glucokinase in a high carb diet) & pancreas b. High Vmax allows liver to effectively remove excess glucose, and minimize hyperglycemia after eating c. High BGL Increases glucose uptake by the liver; allows glucose to be stored as glycogen; BGL exiting liver is lower than entering
i. In liver, insulin stimulates gene transcription and synthesis of glucokinase

d. Not inhibited by G6P (dont want it to be product inhibited); want liver to be able to continue in high glucose conditions

e. Acts as part of glucose sensor system in pancreatic islet -cells (which secrete
insulin in response to high BGL to regulate glucose metabolism in liver & other tissues by mechanisms that will lower the BGL)

i. High BGL high ATP shuts off potassium channel insulin release
1. High BGL 2. Activate glucokinase 3. Beta cell make more ATP
a. Which regulates the ATP-K channel that maintains cell polarity

4. Depolarizes the cell 5. Calcium influx 6. Release of insulin

18. Diabetes a. Type I diabetic i. Higher resting glucose concentration ii. Higher peaks in plasma glucose after meal iii. Return to baseline slower iv. Not producing insulin necessary to control blood sugar b. Type II i. Have the pancreatic islet -cells to make insulin ii. Maturity onset diabetes of the young (MODY): glucokinase gene or transcriptional regulation factor mutation;
1. Is based on a single mutation in one of several genes, so this class of type II is also called autosomal dominant type II diabetes

2. Not enough glucokinase causes -cells to not respond normally to ingested glucose, so they do not secrete insulin normally
3. Mild to moderate hyperglycemia

4.

Is not associated with obesity or high blood lipid levels

INsulIN stimulates 2 things to go IN 2 cells: Potassium and Glucose. "In the Phasted State, Phosphorylate": The phosphorylation cascade becomes active when blood glucose is low

15. THE METABOLISM OF GLUCOSE: GLYCOLYSIS

a. b.

Glucose to Pyruvate (aerobic) or Lactate (anaerobic) yielding 2ATP/mole glucose Eleven enzymatic reactions; all the enzymes are present in the soluble cytoplasm of the cell

i. Hexokinase 1. Input: 2 ATP/ 1 mole glucose

2. Product: Glucose-6-Phosphate + ADP

ii.

3. Irreversible Phosphohexose isomerase

1. 2. Input: Glucose-6-Phosphate Product: Fructose-6-Phosphate

iii. Phosphofructokinase 1 (PFK 1) 1. Input: Fructose-6-Phosphate + ATP

2. Output: Fructose-1,6-bisP

3.

Inhibited by excess ATP, PEP, Citrate, glucagon

a. Glucagon shuts off the kinase activity of PFK2. This reverses any synthesis of F-2,6-BP from F6P and thus inhibits PFK1 activity

4. Relieved by AMP, ADP, Pi a. Fructose-2,6-bisphosphate is an allosteric activator of PFK1

b. When PFK-2/F-2,6-bisphosphatase is phosphorylated (P*) by Protein kinase A in muscle, PFK-2 is activated and F-2,6-bisphosphatase is inhibited. The concentration of Fructose-2,6-bisphosphate is raised so glycolysis is activated Dephosphorylation of PFK-2 drives glycolysis

c.

PFK1 deficiency: exercise intolerance in skeletal muscle iv. Aldolase

1. 2. Input: Fructose-1,6-bisP Product: two triose-phosphate units a. Glyceraldehyde-3-P substrate for next step of glycolysis b. Dihydroxyacetone-P a. b. Input: dihydroxyacetone-P Product: glyceraldehyde-3-P

5. 6.

*First step committed uniquely to glycolysis

3. Possible next step: Triose phosphate isomerase (TIM) c. Pathway only favored when product is removed v. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH)
1.
2.

Input: glyceraldehyde-3-P + NAD+ + Pi

Process: oxidation a. Electrons are accepted by NAD+ coenzyme b. Reaction requires Pi for formation of high energy phosphate bond (at C1) which allows transfer of the phosphate to ADP

3.

Product: 1,3-bisphosphoglycerate + NADH + H+ a. Binds 4 molecules NAD+ but it becomes harder to add each successive molecule

4. Enzyme displays negative cooperativity

b. Buffers the enzyme activity from changes in substrate concentration; enzyme resists the loss of NAD+ that occurs when glycolysis is happening at high rates as in anaerobic muscle
c. Enzyme is sensitive to a much wider range of [S] since it doesnt get saturated immediately

vi. Phosphoglycerate Kinase (PGK)

1. 2. 1. 2. 1. 2. 3. 1. Input: 1,2-bisphosphoglyerate + ADP Product: 3-phosphoglycerate + ATP Input: 3-phosphoglycerate Product: 2-phosphoglycerate Input: 2-phosphoglycerate Process: removal of water with creation of high energy phosphate bond

vii. Phosphoglycerate Mutase (PGM) viii. Enolase a. Strongly inhibited by fluoride

Product: Phosphoenolpyruvate Input: Phosphoenolpyruvate + ADP

ix. Pyruvate Kinase 2. Irreversible reaction 3. Stimulated by fructose-1,6-bisphosphate (feed forward stimulation) 4. Inhibited by: alanine, NADH, ATP, fatty acids, and succinyl-CoA
5. Product: Pyruvate + ATP

6. PK deficiency causes hemolytic anemia

x. Next step happens under anaerobic conditions: Lactate dehydrogenase

1. 2. 2.

3. This step is required for regeneration of NAD+

There is a net yield of 2ATP in the conversion of one glucose to 2 lactates a. One ATP is used in each of the hexokinase and phosphofructokinase reactions b. 2ATPs per glucose are generated in each of the 3-phosphoglycerate kinase & pyruvate kinase reactions

Input: Pyruvate + NADH + H+ Product: Lactate + NAD+

3. Poisons can affect glycolysis a. 2-F-Deoxyglucose at hexokinase i. Allows you to use PET to detect places where a lot of glycolysis is going on tumors b. Arsenate affects G3PDH
Pentavalent arsenate 1. Prevents a net gain of ATP from glycolysis but does not stop the process ii. Mercury compounds & trivalent arsenicals 1. React with sulfhydral group and block the glycolytic process i.

c. Fluoride inhibits enolase 4. Nicotinamide Adenine Dinucleotide (NAD+) is derived from B-vitamin niacin a. Pellagra disease i. Niacin deficiency ii. Poor growth, weight loss, dermatitis, diarrhea, mental disturbances (3 Ds)
5. The reduced coenzyme (NADH) is generated by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and must be recycled (reduced) to NAD+ via lactate dehydrogenase for glycolysis to continue
CH3CH2> CH3CH2OH> CH3CH=O> CH3CH2COOH MORE REDUCED MORE OXIDIZED

6. Irreversible steps are sites of regulation and are inhibited under resting conditions a. Hexokinase b. Pyruvate Kinase i. Shut off in liver under starvation conditions c. Phosphofructokinase i. Actual rate of PFK-1 is determined by the amount of a positive regulator that relieves allosteric inhibition (Fructose-2,6bisphosphate) 1. F-2,6-BP is made from F6P by PFK-II ii. PFK2 can be regulated by hormones such as insulin & glucagon
1. Phosphatase (Phosphorylated form)

2. PFK2 Synthetase (dephosphorylated form) a. Insulin drives dephosphorylation of PFK2 activated PFK2 makes more F2,6BP to positively regulate PFKI to go through the cycle and increase glycolysis
Glycolysis - (PFK 1 -),

F2,6BP

ATP

ADP PFK2-OPO3=

F2,6BP

PFK2

F6P

Pi

F6P

Insulin

Glycolysis (PFK 1 ),

Fructose-2,6-Bisphosphate Is Made From F6P By a Second Phosphofructokinases ( PFK-II )

+ R=O

Pi

Fructose-6-phosphate PFK 2

Allosteric Regulation Pi
Phosphatase I

Phosphorylation Regulation Inhibitor

ATP

PFK 1

Phosphatase II

Regulated Enzyme

Activator

phosphate Hexokinase Glucose-6-P Phosphofructokinase I FructoseATP Stimulator of glycolysis Substrate for glycolysis 2,6-bisP Citrate Fig. 5. The role of pyridine coenzyme (NAD+ and NADH) in electron transfer ADP, Fatty Acids (oxidation/reduction) AMP PEP Pi Pellagra was Glucagon prolonged deficiency of the vitamin is lethal. widespread in prison and orphanage populationsPyruvate early in the Kinase 20 th century before its cause was understood. Inactive when High carb FructoseATP, NADH The glyceraldehyde-3-phosphate dehydrogenase reaction provides one example of how phosphorylated & insulin 1,6-bisP Alanine NAD+ can be used for oxidation. Alcohol dehydrogenase, which converts ethanol to (fructoseFatty acids acetaldehyde, is another. In cells the NAD+/NADH ratio is usually high to support biological 2,6-bisP) oxidation. Lactate dehydrogenase provides an example SuccinylCoA where NADH is used for reduction. High carb Glucokinase High IV. PYRUVATE HAS SEVERAL ALTERNATIVE METABOLIC PATHS & insulin BGL 7. Connecting glycolysis with metabolism of pentoses, lipids and amino acids Pyruvate can serve several metabolic functions. When it is used for reactions other than

Fructose-2,6-bis phosphate

Fructose-1,6-bis

Induced Enzyme Regulation Functional State Induced by

lactate formation, the NAD + required for glycolysis must be regenerated from NADH by other
GLYCOLYSIS PROVIDES PRODUCTS THAT ARE USED FOR IMPORTANT PURPOSES.
GLUCOSE

NUCLEOTIDES GLYCOGEN GLUCOSE-6-P PENTOSE-5-P

Figure 6. Metabolic fates of pyruvate

ATP LACTATE

NADPH LIPIDS

PYRUVATE

AMINO ACIDS

means (Refer to the chapter on Oxidative Phosphorylation). TCA CYCLE,CO , H O The reversible interconversions of lactate, alanine and pyruvate occur in the cytoplasm. In metabolic diseases that cause an increased concentration of any one of these substances, the levels of all three may rise together because they are in equilibrium with each other. Intermediate Can bemembrane. used for: Pyruvate enters mitochondria via a carrier protein in the mitochondrial inner The conversions ofGlucose-6-phosphate pyruvate to oxaloacetate and acetyl CoA occur in mitochondria and are Glycogen, polysaccharides, glycoproteins, irreversible. pentoses, NADPH
2 2

Fructose-6-phosphate &8 glyceradldehyde-3-phosphate Dihydroxyacetone-phosphate 3-phosphoglycerate 1,3-bisphosphoglycerate Pyruvate

Pentoses Glycerol phosphate phosphatidic acid neural fat & phospholipids Serine 2,3-bisphosphoglycerate (BPG) Amino Acids (Alanine) Acetyl CoA fatty acids, cholesterol, steroid hormones

8. Tissue-Specific Differences Cell/ Tissue Erythrocytes Heart muscle, Brain Skeletal muscle

Major ATP Source Glycolysis only Oxidative metabolism, under all conditions Oxidative metabolism, at rest; both oxidative metabolism & glycolysis during exercise

a. RBCs lack mitochondria and depend entirely on anaerobic glycolysis for energy i. Converts pyruvate to lactate which is transported to liver for resynthesis to glucose
1. Provides the cell with a mechanism for the oxidation of NADH (produced during the G3PDH reaction) to NAD+ which occurs during the LDH catalyzed reaction so glycolysis can continue

ii. Hemolytic anemia results from genetic enzyme defects b. Skeletal muscle i. Pyruvate formed enters mitochondria for oxidative metabolism
ii. During hard exercise, the rate of glycolysis in skeletal muscle increases greatly and more pyruvate is produced than can be oxidized in mitochondria

iii. Pyruvate is converted to lactate, regenerating NAD+ in cytoplasm and ATP for the working muscle iv. Lactate is transported to the liver for resynthesis c. Cardiac muscle i. Lactate dehydrogenase is inhibited as pyruvate concentration increases inhibits conversion of pyruvate to lactate; helps direct pyruvate into oxidative metabolism
1. The electrons of cytoplasmic NADH are transferred to mitochondrial carriers of the oxidative phosphorylation pathway generating a continuous pool of cytoplasmic NAD+

Regulation of glycolysis in RBC

Enzyme Hexokinase Phosphofructokinase I Pyruvate kinase Stimulating Regulator (indicative of low energy state) Low G6P Low ATP High AMP High F-1,6-BP

16. OTHER SUGARS (FRUCTOSE, MANNOSE & GALACTOSE) & ALCOHOL a. Sorbitol: can interconvert fructose & glucose
Aldol reductase 1. Input: glucose + NADPH 2. Product: Sorbitol + NADP ii. Sorbitol dehydrogenase 1. Input: sorbitol + NADP i.

2. Product: fructose + NADPH

b. Fructose: i. Fructokinase
1. 2. Input: fructose + ATP Product: fructose-1-phosphate a. Hexokinase can act on fructose to make fructose-6-phosphate but normally the enzyme has a much higher affinity for glucose

3. Essential fructosuria a. Deficient fructokinase b. Fructose levels elevated in blood & fructose appears in urine c. Hexokinases can phosphorylate fructose when the concentrations get too high (back up system)
d. Not associated with clinical abnormalities because there is no accumulation of F1P or ATP depletion

ii. Aldolase type B: cleaves F1P (and also F-1,6-bP) into two trioses 1. One is phosphorylated (dihydroxyacetone-phosphate) a. Triosephosphate isomerse: DHAP G3P glycolysis 2. Glyceraldehyde kinase: glyceraldehyde G3P glycolysis 3. Hereditary Fructose Intolerance a. Aldolase B deficiency i. Sensitivity to dietary fructose, sucrose, sorbitol
1. eating a large bowl of fructose can give temporary symptoms

ii. Accumulate fructose-1-phosphate prevent glycogen breakdown & glucose synthesis iii. Major medical problem; Sweating, trembling, dizziness, nausea, coma; Infants: abdominal distension, poor growth, liver enlargement iv. Does not affect aldolase A or C c. Toxicity of fructose-1-phosphate i. Accumulation is associated with depletion of ATP and Pi ii. Changes in blood concentrations of metabolites 1. Fall in glucose: F1P inhibits release of glucose from glycogen 2. Fall in free phosphorus: Pi is used in re-synthesis of ATP; phosphorus becomes trapped in F1P 3. Rise in uric acid: from increased synthesis & breakdown of purine nucleotides

d. Mannose: i. Hexokinase
1. 2. 1. 2. Input: Mannose Product: Mannose-6-phosphate Input: Mannose-6-phosphate Product: fructose-6-phosphate glycolysis

ii. Phosphomannose isomerase e. Galactose: (lactose = glucose + galactose) i. (1st) Galactokinase 1. Input: galactose 2. Product: galactose-1-phosphate a. Cell must make Uridine diphosphoglucose to use this 3. Galactokinase Deficiency 1. High levels of blood galactose 2. Some is reduced to galactitol (via aldol reductase) which can lead to development of cataracts ii. UDP-glucose pyrophosphorylase 1. Input: glucose-1-phosphate + uridine triphosphate (UTP) + Pi 2. Product: uridine diphosphoglucose (UDP-glucose) nd iii. (2 ) Galactose-1-phosphate uridyltransferase (UDP-Glc)
a. Input: UDP-glucose + galactose-1-phosphate b. Product: UDP-galactose + glucose-1-phosphate

iv. Galactose-1-phosphate uridyltransferase deficiency 1. Classic Galactosemia; Autosomal recessive 2. Infant: cannot utilize galactose; must exclude from diet a. galactose-1-phosphate accumulates; some is made into galactitol-1-phosphate both toxic b. Lack of growth, vomiting, dehydration, jaundice, eventually developmental retardation and cataracts 3. Can still synthesize galactose containing glycoproteins & glycolipids from glucose via UDP-glucose formation and epimerase activity to form UDP-galactose GALIPUT: Galactose 1 Phosphate Uridyl Transferase. 4. UDP-galactose-4-epimerase a. Input: UDP-galactose + NAD+ b. Product: UDP-glucose + NADH c. Allows galactose and N-acetylgalactosamine containing glycoproteins and glycolipids to be synthesized from glucose even when no galactose is provided in the diet
d. UDP-galactose-4-epimerase deficiency i. Rare, recessive ii. High levels of blood galactose-1-phosphate

v.

(3rd) Phosphoglucomutase 1. Input: glucose-1-phosphate 2. Process: isomerization (~phosphoglycerate mutase reaction of glycolysis) 3. Product: glucose-6-phosphate glycolysis

1. Alcohol Metabolism f. Oxidation of ethanol to acetaldehyde: three different pathways:

i. Cytosol 1. Alcohol dehydrogenase (ADH) & NAD+ 2. ADH has broad specificity 3. There are 5 different ADH genes whose polymorphisms contribute to the ability of people to oxidize ethyl alcohol Smooth ER 1. Microsomal ethanol oxidizing system (MEOS) 2. Becomes important in high alcohol intake 3. Alcohol can also interfere with cytochrome P450 Peroxisomes 1. Catalase

Alcohol Is Converted to Acetaldehyde and the Acetic Acid

ETHANOL
NADP+ NAD+

(Adaptive)

MEOS (CYTP450) NADPH + H+

ALCOHOL DEHYDROGENASE NADH + H+

ii.

ACETALDEHYDE ACETATE Acidosis AcetylCoA

iii.

g. Acetaldehyde i. Reacts with amino groups & proteins (cross-linking) ii. Inhibits mitochondrial functions 1. Feedback loop: high acetaldehyde impairs mito function leading to accumulation of more acetaldehyde
iii. Aldehyde dehydrogenase (ALDH) 1. Input: acetaldehyde + NAD+ 2. Product: acetate + NADH + H+ 3. Two forms of the enzyme; ALDH2 is most important & gene occurs in two polymorphic forms a. Wild type: active enzyme b. Second form: inactive i. Dominant allele; reduced ability to oxidize acetaldehyde vasodilation, facial flushing iv. AcetylCoA Synthetase 1. Input: acetate + ATP + CoA

2. 3.

Product: acetylCoA + AMP + PPi Allows recovery of small amount of energy

PRODUCTION OF NADH INHIBITS GLUCONEOGENESIS

LACTIC ACIDOSIS

LACTATE NADH NAD+ MALATE

PYRUVATE

OXALOACETATE

GLUCONEOGENESIS

PRODUCTION OF NADH STIMULATES FAT PRODUCTION Alcoholics are often vitamin deficient: Thiamine deficiency causing ophthalmoplegia, gait difficulties and confusion

h. Acidosis i. Excess acetate appears in the blood ii. Deficiency of NAD+ leads to increased conversion of pyruvate to lactate by lactate dehydrogenase i. Gluconeogenesis i. Demand for pyruvate reduces its availability for gluconeogenesis ii. Ratio of NADH : NAD+ goes up; reduces amount of pyruvate; shut downs down gluconeogenesis
iii. Lack of gluconeogenesis combined with poor nutrition can lead to hypoglycemia

j. Triglycerides/ Fat production i. Excess NADH supports production of glycerol-3-phosphate from dihydroxyacetone phosphate ii. Depletion of NAD+ impairs the ability to oxidize fatty acids iii. Results in increased triglycerides that can be either secreted into the plasma or deposited into the liver k. Vitamin deficiency
i. Pyridoxine or folate deficiencies hemoatologic problems; neurological problems

ii. Wernicke-Korsakoff syndrome (ataxia, mental disturbance, uncoordinated eye movements) 1. An alcoholic can present with these symptoms due to a thiamine deficiency l. Poisons i. Methanol (Moonshine)
1. 2. 3. 1. Metabolized by same enzymes that metabolize ethanol Alcohol dehydrogenase activity on it produces formaldehyde and formic acid death Supplementing with ethanol can reverse condition Ethylene glycol is metabolized to glycoaldehyde by alcohol dehydrogenase a. Then to glycolic acid by aldehyde dehydrogenase b. Results in severe acidosis

ii. Antifreeze

17. OXIDATIVE METABOLISM - THE TRICARBOXYLIC ACID CYCLE 1. TCA: Mitochondria Matrix a. Inner membrane contains a carrier protein that allows pyruvate to enter the matrix 2. Citrate is exported from the mitochondria to allow fat synthesis & inhibit glycolysis a. Acetyl-CoA is converted to citrate i. There is no way to get acetyl-CoA directly out of mitochondria; must convert to citrate first 3. CAN cross mitochondrial membrane a. Pyruvate; citrate; isocitrate; -ketoglutarate; succinate; fumarate; malate; ATP, ADP, Pi 4. CAN NOT cross mitochondrial membrane a. Acetyl CoA; Oxaloacetate; NAD+, NADH
AcetylCoA For the TCA Cycle Can Be Derived from Other Products

CO2 + Reduced
coenzymes TCA

ATP + H2O

Pyruvate Fatty acids Amino acids

Acetyl-CoA

Fatty acids Cholesterol, steroids

Ketone bodies AcetylCoA Can Oxidized Or Converted to Other Products

5. Pyruvate Dehydrogenase a. Substrate: Pyruvate (3C) + CoA + NAD+ b. Product: Acetyl(2C)-CoA + NADH + H+ + CO2 c. Regulation: i. Not reversible ii. Inhibited by 1. Acetyl CoA; NADH, ATP a. Activates the kinase which phosphorylates pyruvate dehydrogenase into its INACTIVE form b. Kinase: Phosphorylated = ACTIVE PDH INACTIVE c. Kinase Inhibited by products (pyruvate, NAD+, CoASH); dichloroacetate iii. Activated by

Pyruvate Dehydrogenase Is Regulated by Feedback Inhibition and Phosphorylation

Dichloroacetate
Pyruvate dehydrogenase inactive

Pi

ADP

Mg2+ Kinase

Mg2+ Ca2+ Phosphatase

+
Insulin +

+ +
Pyruvate dehydrogenase active

+ ATP

Pi

Pyruvate CoASH NAD+

Acetyl CoA

CO2 NADH

1. Insulin, Mg2+, Ca2+, pyruvate, NAD+, CoASH, Dichloroacetate a. Phosphatase activated by Ca2+ (in exercising muscle) & insulin (well-fed state) means you want to do a lot of glycolysis & TCA cycle b. Phosphatase: Dephosphosphorylated = ACTIVE PDH ACTIVE

The Metabolic Pathways for Glucose Require Coenzymes: CoA/Acetyl CoA: Transfers Acyl Groups Thiamine Pyrophosphate: Decarboxylation and 2 Carbon Transfer Lipoic Acid: Acetyl Transfer Biotin: Carboxylation NAD+/NADH: electron transfer for oxidation NADP+/NADPH electron transfer for reduction FAD/FADH2 electron transfer for oxidation/reduction
Pyruvate dehydrogenase alone has 5 Coenzymes involved: CoA, TPP, Lipoate, FAD, NAD+

Deciencies produce syndromes: niacin and pellagra, thiamine and beri beri or Wernicke-Korsakoff.

d. Pyruvate dehydrogenase i. Thiamine Pyrophosphate (TPP): coenzyme on E1 1. Derived from Vitamin B1 (thiamine)
2. Bings pyruvate on thiazole ring; then CO2 is released, leaved 2 Carbons on the ring

ii. iii.
iv. v.

3. Deficiency leads to confusion, irritability, weight loss, edema, heart failure: Wernickes Encepalopathy (WernickeKorsakoff), beri-beri (wet or dry) Lipoate: active arm on E2
1. Transfers acetyl group to CoA, and electrons to a coenzyme (FAD) on E3

2. Target for Trivalent arsenic (AsO2-) (arsenic poisoning) FAD: Flavin Adenine Dinucleotide: Coenzyme on E3 1. Derived from Vitamin B2 (riboflavin)
Coenzyme A/ Acetyl Coenzyme A NAD+

e. Leighs Disease i. Deficiency in PDH

ii. iii. Degeneration of CNS; Neurological affects: loss of head control, motor skills, seizures; Lactic acidosis can lead to impairment of respiratory & kidney function Prognosis: poor

iv.

Treatment: thiamine or Vitamin B1; high fat, low carb diet; oral sodium bicarbonate/citrate 1. Experimental protocols: using dichloroacetate (reduces inhibitory phosphorylation of PDH)

f.

Succinate dehydrogenase and fumarase catalyze sequential steps in the TCA cycle. Homozygous mutations in either gene can result in severe neurological impairment. Germline heterozygous mutations of succinate dehydrogenase are

phaeochromocytoma and paraganglioma. Mutations in fumarase cause a predisposition to cutaneous

and uterine leiomyomas, as well as to kidney cancers.

6. TCA Cycle: Generates reduced coenzymes (FADH2 & NADH) that can be treated by ox-phos to generate energy; also generate one GTP a. Citrate synthase i. OAA + Acetyl CoA citrate ii. Irreversible; Regulated iii. Inhibited by: NADH; succinyl CoA iv. Rate increases as [OAA] & [Acetyl CoA] increase b. Aconitase i. Citrate isocitrate
ii. Although citrate appears to be a symmetrical molecule, its two ends are handled asymmetrically because it can bind on only one way in the asymmetric binding site of the enzyme

c. Isocitrate dehydrogenase i. Isocitrate + NAD+ -ketoglutarate + NADH + H+ + CO2 d. -ketoglutarate dehydrogenase i. -ketoglutarate + NAD+ Succinyl-CoA + NADH + H+
ii. Five coenzymes: TPP, lipoate, CoASH, FAH, NAD+

1.

~pyruvate dehydrogenase

iii.
iv.

Inhibited by: NADH; ATP; succinyl CoA

Lipoate is the target of arsenic

e. Succinyl CoA synthetase i. Succinyl-CoA + GDP + Pi Succinate + GTP

ii. Succinate is symmetrical

f. Succinic acid dehydrogenase i. Succinate + FAD Fumarate + FADH2 ii. Inhibited by: OAA iii. Activated by: ATP; Pi; succinate g. Fumarase i. Fumarate L-Malate h. Malate dehydrogenase i. L-Malate + NAD+ Oxaloacetate (OAA) + NADH

Our City Is Kept Safe And Sound From Malice

AcCoA
COOC=O H-C-H COO-

The TCA Cycle Is A Connector For CHO, Fat & Amino Acid Metabolism
CoA
COOCH2 HO- C- COOCH2 COOCITRATE

The 30,000 Ft View

Condense

NADH H+ Oxidize

Remove, Add H2O

OXALOACETATE

GLUCOSE
COOCH2 H- C- COOHO- CH COO-

COOH- C-OH H- C- H COO-

MALATE ISOCITRATE

Amino Acid

OXALOACETATE

CITRATE

Add H2O
COOH- C FUMARATE H-C COOCOOaKETOGLUTARATE CH2 C-H2 C=O COOSUCCINYLCoA COOH- C-H H- C-H C=O SCoA

Oxidize CO2 NADH H+

MALATE

ISOCITRATE

Amino Acids

TCA Cycle
FUMARATE aKETOGLUTARATE SUCCINATE SUCCINYLCoA

SUCCINATE

FADH2 Oxidize

COOH- C-H H- C-H COO-

Oxidize CO2 NADH H+

GDP
GTP

Thiokinase + Pi

Can I Keep Selling Sex For Money Officer?

AcCoA
COOCH2 HO- C- COOH- C-H Citrate Synthase CH2 COOCOOCOOC=O

Can either drain intermediates from the cycle or pump the cycle up by supplying them (anaplerotic reactions, e.g. pyruvate carboxylase)

CoA

Condense

Regulators of the TCA Cycle

Aconitase: Stereoselective & Inhibited By Fluoroacetate
COOCH2 H- C- COOHO- CH COOATP.AcCoA.NADH,fatty acids [NADH,SuccCoA,citrate, ATP] AMP, CoA, NAD+ , Ca++ [ADP]

NADH H+ Oxidize (Malate dehydrogenase)

COOH- C-OH H- C- H COO-

OXALOACETATE

CITRATE MALATE ISOCITRATE

NADH

Fumarase is stereoselective

COOH- C FUMARATE H- C COO-

SUCCINATE

COOCH2 aKETOGLUTARATE C-H2 C=O COOSUCCINYLCoA COOH- C-H H- C-H C=O SCoA

Oxidize CO2 NADH H+ (Isocitrate dehydrogenase)

ADP, Ca++

DG -4.2kcal

NADH , ATP

COOH- C-H (Succinic acid dehydrogenase) H- C-H COO-

FADH2

Oxidize

CO2 NADH H+ Oxidize (a-Ketoglutarate dehydrogenase, coenzymes as PDH, inhibited by arsenic,

DG -10.5kcal

Ca++ NADH,SuccCOA

GDP
GTP

Succinyl CoA Synthetase (Thiokinase) + Pi ATP

Regulated Enzyme Citrate synthase

Activator (low energy state)

Isocitrate dehydrogenase

Isocitrate (insulin regulation

results in citrate buildup)

-ketoglutarate dehydrogenase

ADP, AMP NAD+ Ca++ Ca++

Inhibitor(high energy state) NADH ATP Succinyl CoA NADH ATP

NADH Succinyl CoA

7. Reduced coenzymes come from: a. NADH: Pyruvate dehydrogenase; Isocitrate dehydrogenase; -ketoglutarate dehydrogenase; Malate dehydrogenase ANAPLEROTIC PATHWAYS b. FADH2: Succinate dehydrogenase 8. GTP comes from: Succinyl-CoA synthase Pyruvate PEP Carboxylase 9. Anaplerotic pathways Malic Enzyme Carboxykinase a. Need to replenish the intermediates
GLUCOSE PHOSPHOENOLPYRUVATE PYRUVATE AcCOA

i. ii. iii. iv. v. a. b. c. d. e. f. g.

Glutamate dehydrogenase: -ketoglutarate Malic enzyme: Pyruvate Malate PEP carboxykinase: PEP OAA Pyruvate carboxylase: Pyruvate OAA Transminase: Amino acids OAA

(D)

OXALOACETATE

CITRATE

Transaminase

MALATE

A,P,Y

FUMARATE

aKETOGLUTARATE

10. TCA Cycle Intermediate links

All intermediates except malate, isocitrate, and citrate (M.I.C.) can be turned into amino acids Acetyl CoA fatty acids & steroid biosynthesis Citrate fatty acids/ sterol synthesis (cytoplasm) -ketoglutarate glutamate amino acid purines Succinyl-CoA heme Malate glucose synthesis (cytoplasm) Fumarate urea cycle (tyrosine & phenylalanine breakdown)

SUCCINYL-CoA Glutamate GLUTAMATE (E)

Dehydrogenase

I,M,V Odd-chain FA

Alternative Fates of Pyruvate

Glucose Glycolysis Pyruvate

h. Oxaloacetate i. OAA formed by pyruvate carboxylase pyruvate ii. asparate amino acid purines/ pyrimidines iii. Interconverted with malate & exported to cytoplasm to act as substrates for gluconeogenesis
1. 2. Convert to malate generate NADH in cytoplasm Or can convert to asparate

Transamination
nine Ala

Reduction

Carboxylation

Lacta te Oxidative decarboxylation

Oxaloacetate

Acetyl CoA

TCA CYCLE

18. THE PENTOSE PHOSPHATE PATHWAY

1.

Using G6P for the biosynthesis of pentoses or formation of NADPH a. Ribose-5-phosphate is needed for nucleotide synthesis. b. NADPH is need to reduce glutathione, to synthesize fatty acids, Nitrous Oxide, and steroids/sterols, to detoxify (cytochrome P450) c. In most tissues 80-90% of glucose oxidation is by Glycolysis the rest 10-20% is by PPP i. 5-10% of liver glucose metabolism; More in adipocytes

2. Two stages: a. Oxidation i. Glucose-6-phosphate dehydrogenase

1. G6P + NADP+ 6-phosphoglucono--lactone + NADPH + H+

2. Committed step; rate limiting

3. Coenzyme: NADP+

4. Activated by: Insulin 5. Inhibited: Allosterically by NADPH (potent competitive inhibitor) b. As NADPH is depleted more NADP+ is formed c. High NADP+ stimulates Glucose 6P dehydrogenase and NADPH production 6. G6PDH deficiency a. Precipitation of hemoglobin occurs due to disulfide bond formation between Hb molecules
b. Extremely common; May present with: Dark colored urine; Low RBC count; RBC with inclusion bodies; Elevated reticulocyte count; Low hemoglobin; Elevated serum bilirubin i. Class I: <2% enzyme activity very severe ii. Class III (A-): 10-50% enzyme activity modest 1. Patient with 10% of normal activity have enough to generate NADPH under normal conditions. ii. Lactonase 1. 6-phosphoglucono--lactone + H2O 6-phosphogluconate a. Has a lower Ki than Km of NADP+ when present at higher concentrations

c. Clinical disease is related to degree of defect

iii. 6-phosphogluconate dehydrogenase 1. 6-phosphogluconate + NADP+ Ribulose-5-P + NADPH

2. 2nd formation of NADPH molecule

b. Isomerization & Epimerization i. Ribulose-5-Isomerase (phosphopentose isomerase) 1. Ribulose-5-P Ribose-5-P ii. Ribulose-5-Epimerase (phosphopentose epimerase) 1. Ribulose-5-P REVERSIBLE INTERCONVERSIONS OF PENTOSE-P Xylulose-5-P AND GLYCOLYTIC INTERMEDIATES 3. Transketolase Transketolase a. Xylulose-5-P Ribose-5-P Xylulose + Ribose Sedoheptulose + Glyceraldehyde 5-P 5-P 7-P 3-P b. Transfers 2 Carbon Units Using Thiamine (TPP) Transaldolase Sedoheptulose + Glyceraldehyde Erythrose + Fructose c. Wernicke-Korsakoff Syndrome 7-P 3-P 4-P 6-P 4. Transaldolase a. Transfers 3 carbon units Xylulose Erythrose Transketolase Glyceraldehyde + Fructose
5-P

4-P

3-P

6-P

3 Pentose-P

1 Triose-P + 2 Hexose-P

5.

Four Modes of the PP Pathway: a. Balanced need for NADPH and R5P b. Need R5P (more than NADPH) i. Rapidly dividing cells c. Much more NADPH than R5P is needed i. Synthesis of fatty acids in adipose tissue d. Both NADPH and ATP are required; R5P can be converted to pyruvate which can be oxidized to generate more ATP i. Biosynthesis
DIFFERENT MODES OF THE PP PATHWAY
2NADP+ 2NADPH

DIFFERENT MODES OF THE PP PATHWAY

2NADP+ 2NADPH

DIFFERENT MODES OF THE PP PATHWAY

2NADP+ 2NADPH

DIFFERENT MODES OF THE PP PATHWAY

2NADP+ 2NADPH

GLUC-6-P
CO2

RIBULOSE-5-P

GLUC-6-P
CO2

RIBULOSE-5-P

GLUC-6-P
CO2

RIBULOSE-5-P

GLUC-6-P
CO2

RIBULOSE-5-P

FRUC-6-P

RIBOSE-5-P

FRUC-6-P

RIBOSE-5-P

FRUC-6-P

RIBOSE-5-P (1,2)

FRUC-6-P

RIBOSE-5-P

FRUC-1,6-BP

1: NADPH ~RIBOSE

FRUC-1,6-BP

FRUC-1,6-BP

FRUC-1,6-BP

GLY3-P

GLY3-P

2: RIBOSE > NADPH

GLY3-P

GLY3-P

4: NADPH > RIBOSE

3: NADPH >> RIBOSE, CO2

PYRUVATE

PYRUVATE

PYRUVATE

PYRUVATE

6. Net result: Oxidation of G6P, a 6 carbon sugar, into a 5 carbon sugar

i. In turn, 3 moles of 5 carbon sugar are converted back into two moles of 6 carbon sugars and one mole of 3 carbon sugar

1. The 6 carbon sugars can be recycled into the pathway in the form of G6P, generating more NADPH. 2. The 3 carbon sugar generated is glyceraldehyde-3-phsphate which can be shunted to glycolysis and oxidized to pyruvate. a. Alternatively, it can be utilized by the gluconeogenic enzymes to generate more 6 carbon sugars (fructose-6phosphate or glucose-6-phosphate).

7. Cells have a high NADPH versus NAD+ (opposite of NAD & NADH); Two oxidation-reduction systems exist side by side with opposite polarities; both systems utilize the same active site a. NAD+: coenzyme of oxidation in mitochondria & nucleus i. Gets reduced to NADH ii. Glycolysis oxidation reactions (usually have with dehydrogenase enzyme reactions) iii. Want cell to have high NAD and low NADH b. NAPH: coenzyme of reduction in cytosol i. Gets oxidized to NADP ii. Making fat (unsaturated to saturated double bone) iii. Want high NADPH, low NADP (would drive reactions the opposite way)

8. Oxidative Damage a. superoxide dismutase converts superoxide to hydrogen peroxide b. Glutathione (GSH) is required for reduction of peroxides by peroxidase (selenium containing enzyme)
i. Also responsible for maintaining the intracellular environment in a reduced state so that disulfide bonds in proteins stay reduced

c. NADPH produced by glucose-6-P dehydrogenase maintains the supply of reduced glutathione needed to destroy peroxide by regenerating the reduced form of GSH
NADPH produced by glucose-6-P dehydrogenase maintains the supply of reduced glutathione needed to destroy peroxide

G6P Dehydrogenase

GSH peroxidase

G-6-P 6-PGluconate

NADP+

G-SH

H2O2

NADPH G-S-S-G + H+ GSSG reductase

H2O

d.

Defect means you cant detoxify peroxide and damages RBCs i. RBCs are more dependent on G6PDH than other cells that have alternative pathways to produce NADPH, such as malic enzyme

19. GLUCONEOGENESIS 1. Liver provides 80-90% of glucose; two sources: a. Glycogen breakdown i. Sustains BGL for a few hours after a meal b. Gluconeogenesis i. Sustains BGL for many days in absence of carbohydrate intake
2. Kidneys produce 10-20%, but more on fasting

3. Use 2 pyruvate, 4 ATP, 2 GTP, 2 NADH get ONE molecule of glucose 4. Substrates: a. Lactate (via oxidation of pyruvate) b. Amino acids/ Alanine (via pyruvate, TCA intermediates) c. Glycerol (via glycolysis intermediates; use glycerol backbone of triglycerides to make glucose)
i. Glycerol enters the pathway much later down (at level of DHAP) versus lactate/ pyruvate inhibition of earlier enzymes dont affect as severely

5. Gluconeogenesis goes through multiple (three different) cellular compartments a. Mitochondria cytoplasm ER
b. G6P (end product) is hydrolyzed in ER i. Hexokinase and glucose-6-phosphatase are localized differently

c. OAA needs a transporter to get out of mitochondria i. Conversion to PEP (through the action of the mitochondrial PEPCK) ii. Transamination to aspartate iii. Reduction to malate 1. Requires use of an NADH (will be accumulating in the mitochondrion as the energy charge increase); gets regenerated in cytoplasm when converted back to OAA 6. The four steps that occur in gluconeogenesis but not in glycolysis
Gluconeogenesis Needs to Get Around the 3 Glycolytic Steps Characterized by Large Negative DG
2 Pyruvate + 4ATP + 2GTP + 2NADH +6H2O Glucose + 4ADP + 2GDP + 6Pi + 2NAD+ + 2H+
CO2 Pyruvate Oxaloacetate
Pyruvate

Phosphoenolpyruvate GTP GDP

Hexokinase

Carboxylase

PEPCK (PEP Carboxykinase)

2-PG

Glucose

Glucose-6-phosphate
3-PG ATP 1,3-BisPG Pi G-3-P DHAP ADP

Glucose 6 phosphatase Phosphofructokinase I

Fructose-6-phosphate

Fructose 1,6 bisphosphatase Pyruvate kinase

Fructose-1,6bisphosphate

NAD+

Fructose-1,6-BisPhosphate

Phosphoenolpyruvate
more complicated,

Pyruvate
Pi Glucose
+

Pi
Glucose 6 Phosphatase

Fructose 1,6 Bishosphatase (FBPase)

F-6-P G-6-P

Oxaloacetate made 1st

a. Pyruvate Carboxylase i. Pyruvate + ATP + CO2-Biotin-Enzyme OAA

ii. Carboxylases add HCO3- (i.e. CO2) to substrate

iii.

Coenzyme: Biotin
1. Biotin problems: a. Tremendous injection of egg white (abatin binds biotin) b. Biotinidase deficiency: Biotinidase recovers biotin from proteins being degraded

b. PEP Carboxykinase (PEPCK) i. OAA + GTP P-enolpyruvate (PEP) + CO2 + GTP ii. Cofactor: GTP
iii. iv. Can occur in either cytosol or mitochondria Regulated by glucocorticoids such as cortisol

c. Fructose-1,6-Bisphosphatase i. F-1,6-BP F6P ii. Activated by: Citrate iii. Inhibited by: AMP; F-2,6-BP (low energy levels) iv. *Regulation is the opposite of PFK1 d. Glucose-6-Phosphatase e. Regulation
Regulation of PFK2 Coordinates Glycolysis & Gluconeogenesis

Glucagon
Glycolysis - (PFK 1 -), Gluconeogenesis (FBPase ) F2,6BP ATP

PKA
ADP PFK2-OPO3= F6P F2,6BP

PFK2

F6P

Pi

Insulin

Glycolysis (PFK 1 ), Gluconeogenesis- (FBPase -)

i. ii. iii.

PFK2: enzyme that synthesized F2,6BP which is a potent activator of glycolysis (PFK1) & inhibitor of gluconeogensis (FBPase) Insulin promotes the dephosphorylation Stimulates phosphatase Glucagon stimulates cyclic AMP & phosphorylation of PFK2 F2,6BP hydrolyzed to F6P 1. Glycolysis decreases/ Gluconeogenesis increases (Fbase not inhibited)

7. Glucagon a. Liver: Stimulates gluconeogenesis and glycogenolysis

b. No effect on muscle (no receptor)
Glucagon 29 amino acids 1 polypeptide chain Insulin 51 amino acids 2 polypeptide chains (A = 21, B = 30) Target tissues: liver, adipose skeletal muscle Target metabolism: carbohydrates, lipids proteins

8. Insulin a. Muscle: stimulates glucose uptake

b. Liver: Stimulates glycogenesis

Glucagon Stimulates Gluconeogenesis

Process Glucose uptake (muscle) Gluconeogenesis (liver) Glycogenesis (liver ) Glycogenolysis (liver) Insulin + Glucagon
O (no receptor)

Target tissues: liver, adipose

+ -

+
+

Target metabolism: carbohydrates,lipids

Signal of fasting

Signal of feeding

Metabolic Actions of Insulin and Glucagon

Insulin Fatty acid uptake and release in fat. Glucagon

Stimulates synthesis of triglycerides (TG) from free fatty acids (FFA); inhibits release of FFA from TG.

Stimulates release of FFA from TG.

Insulin Liver glycogen Glucagon

Increases synthesis and thereby glucose uptake and storage.

Stimulates glycogenolysis and glucose release.

Insulin Liver gluconeogenesis Glucagon

Inhibits, saves amino acids.

Stimulates, glucose synthesized and released.

Insulin Glucose uptake, skeletal muscle Glucagon

Stimulates uptake, storage as glycogen and use in energy metabolism.

No receptors, no effect.

Glycogen, skeletal muscle

Insulin

Stimulates synthesis.

Glucagon

No receptors, no effect.

Amino acid uptake

Insulin

Stimulates and is necessary for protein synthesis.

Glucagon

No receptors, no effect.

Brain (hypothalamus)

Insulin

Reduces hunger through hypothalamic regulation.

Glucagon

No effect.

Cycles Between Tissues:

9. Cori Cycle a. RBC or Muscle: Glucose Lactate put into circulation and travels to liver where it is made into glucose and put back into circulation to be used in RBC & muscle cell b. Is an energy loosing deal i. Get 2 ATP in muscle and costs 6 ATP in liver

Cori Cycle

Glucose
Glycolysis 2 ATP

Glucose
Gluconeogenesis

6 ATP

Lactate

Lactate

Red blood cell Muscle cell

Blood

Liver cell

Cycles Between Tissues: Cahill/ Alanine Cycle

Glucose

Glucose

10. Cahill Cycle Glycolysis Gluconeogenesis 2 ATP 6 ATP a. Muscle: Conversion of pyruvate to alanine Pyruvate Pyruvate b. Converted back to pyruvate and then to glucose Alanine Alanine in liver Blood Muscle cell Liver cell 11. Oxidizing alcohol generates excess NADH, depriving liver of gluconeogenic substrates a. Production of NADH by alcohol metabolism blocks gluconeogenesis by converting pyruvate back to OAA rather than lactate; also blocks conversion of malate
Glutamate a-ketoglutarate

20. GLYCOGEN METABOLISM 1. Glycogen metabolism is a story of muscle and liver: a. Muscle (up to ~400g) uses glycogen for its own energy (lacks glucose-6phosphatase) b. Liver (up to ~100g) exports glucose to the system (brain). 2. Short term fix; rapid metabolism a. Amount of glycogen stored is limited b. Long term is gluconeogenesis & fat burning 3. Glucose-6-phosphate a. Stimulates glycogen synthesis & decreases breakdown 4. Fructose-1-phosphate a. Inhibits glycogen breakdown. 5. Insulin leads to a decrease in BGL a. Stimulates glycogen synthase b. Decreases phosphorylase activities 6. Glucagon & epinephrine a. Promote glycogen breakdown and inhibit synthesis. 7. Neuromuscular stimulation a. Activates phosphorylase kinase via acetylcholine receptor and Ca++ to promote breakdown. 8. Breakdown of Glycogen: a. Glycogen phosphorylase cleaves one glucose residue at a time by phosphorolysis from the end of the chain. i. This is the regulated step (McArdles Disease). ii. Product is glucose-1-phosphate b. Debranching enzyme transfers three residues of a branching chain to a different chain and then cleaves the remaining glucose by hydrolysis. i. Product is free glucose c. Ratio of 12:1 of G1P : free glucose d. Glycogen phosphorylase two distinct conformational states: a T (for tense, less active) and R (for relaxed, more active) state; i. Low BGL glucagon Phosphorylate Glycogen phosphorylasea (R/ active state) MORE ACTIVE Breakdown Glycogen

1. Also enhanced by binding of AMP 2. Inhibited by ATP; G6P; Insulin 9. Synthesis of Glycogen: a. Glycogen synthase elongates the chain by one glucose residue at a time from UDPglucose to the 4OH of a glucose at the end of the chain i. Requires a primer which can be made by glycogenin ii. This is the regulated step b. Branching Enzyme transfers a terminal segment of growing chain to form a branch linked a1-6 to a different chain.
i. Synthesis slows down as the particle gets bigger; the amount of glycogen we store is limited

c. Glycogen synthase i. High BGL Insulin activate protein phosphatate Dephosphorylate Glycogen synthase MORE ACTIVE Synthesize Glycogen ii. Low BGL glucagon activate protein kinase Phosphorylate Glycogen synthase Less active
Glycogen phosphorylase and synthase are regulated in a coordinated way
Phosphorylase kinase

Glucagon
P-Glycogen phosphorylase a (more active)

Glycogen phosphorylase b (less active)

Insulin

Phosphorylase phosphatase

Protein kinase

Glucagon
P-Glycogen synthase (less active)

Glycogen synthase (more active)

ATP

ADP

Pi

Insulin

Protein phosphatase

10. Glycogen Storage Diseases

Enzyme

Location Liver Kidney

Glycogen Increased amount Normal structure Large increase Normal structure Increased amount Short outer branches Normal amount Long outer branches Increased amount Normal structure Increased amount Normal structure Increased amount Normal structure

Features Severe hypoglycemia Hepatomegaly Cardiorespiratory Failure

Presentation Lactic acidosis Hypoglycemia Hyperuricemia Hyperlipidemia Hepatomegaly Hypotonia Muscular weakness Heptomegaly (enlarged liver) Hypoglycemia, Hyperlipidemia Short stature

Treatment Constant Glucose infusion Enzyme replacement therapy High Carb High Protein

Type I Von Gierke Type II Pompe Type III Cori

Glucose-6-Phosphatase

-1,4 glucosidase (lysosomal)

Everywhere

Debranching Enzyme

Muscle Liver

Similar to but milder than Type I

Type IV Andersons

Branching Enzyme

Liver

Hepatomegaly Fatal

Type V McArdle Type VI Hers Type VII Tauris

Phosphorylase

Muscle

Exercise problems Cramps

Burgundy colored urine

Phosphorylase

Liver

Similar to but milder than Type I

Hypoglycemia, Hepatomegaly Lactic acidosis Muscle cramps Blood lactate not elevated hemolytic anemia

Carb Supplements

Phosphofructokinase1

Muscle

Like Type V Exercise intolerance

Andersons is only disease of glycogen synthesis Glycogen storage: names of types I through VI "Viagra Pills Cause A Major Hardon": Von Gierke's Pompe's Cori's Anderson's McArdle's Her's Very Poor Carbohydrate Metabolism (V,P,C,M) of Hers (H) HEpatic glycogen phosphorylase deficiency ABCD:

Anderson's=Branching enzyme. Cori's=Debranching enzyme. Pompe's disease: type "Police = Po + lys": lysosomal storage disease (alpha 1,4 glucosidase) 11. Glucose-6-Phosphatase deficiency (glycogen storage disease type I) a. Blood sugar concentration i. [BGL] low G6P cant convert to free glucose to be released b. Rate of glycolysis i. High rate of glycolysis due to high [G1P] from glycogen breakdown c. Rate of glycogen synthesis i. Decreased d. Source of free blood glucose i. Break down glycogen 1. Release free glucose 2. Rest of G1P (10:1) goes through glycolysis e. Rate of pentose synthesis i. Increase [G6P] Increase PPS ii. Breaking down nucleotides increase uric acid in blood 1. High level of glycolysis & lactic acid f. Accumulation of lipids in liver and blood i. NADPH & substrates (DHAP, citrate, acetyl-CoA, G6P) are still available for FA synthesis g. Development of gout and approaches to control of the consequences of the disease i. Constant supply of glucose