BIOCHEMISTRY

13. THE CONJUGATION OF CARBOHYDRATES TO EACH OTHER & PROTEINS

1. Produces either an α (below) or β-glycoside (above) depending on oxygen positiona. Determines if digestion occurs (as beta in cellulose à cant digest)

2. Polymerization involves activated sugar nucleotide intermediates (e.g. UDP-Galactose)

3. Maltose : two glucoses in an α(1à4) linkage4. Lactose : galactose & glucose in β(1à4) linkage5. Sucrose : glucose & fructose (α-D-glucosyl-β-D-fructoside)

6. Glycogen: homopolymers of glucose; chains of glucose with α(1à4) linkages with closely spaced branching via α(1à6) linkages every 8-10 residues; spherical particles in cytoplasm

7. Starch: homopolymers of glucosea. Amylose : unbranched chain with α(1à4) linkage helical coilb. Amylopectin : α(1à4) linkages but is branches every 12 residues via an

α(1à6) linkage8. Cellulose : Unbranched chain with β(1à4) linkages9. Glycosaminoglycans (GAGs) : long unbranched polysaccharides of repeating anionic

disaccharides that may be sulfated; important part of connective tissuea. 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 cofactorsa. Is for secreted proteins or domains facing extracellularly (except for O-GlcNac)b. 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 timea. Are glycosylated in the Golgib. E.g. blood group substances; salivary musins

13. N-linked glycoproteins have carbohydrate first added as a preassembled 14 sugar blocka. 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 N-acetylglucosamine-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 systema. Recruitment of WBC into areas of infectionb. Leukocyte-adhesion deficiency II

i. Many infections early in life; inability to create pus at wound site; don’t 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. Lysosomes have both endo and exoglycosidasesb. 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. Glycogen storage disease type II2. 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

enzymes1. Normally, N-linked glycoproteins can be modified to generate mannose 6-

phosphate, 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

1. Monosaccharides: glucose, fructose2. Disaccharides

a. Maltose (Glu-Glu)b. Lactose (Gal-Glu)c. Sucrose (Fru-Glu)

3. Polysaccharidesa. Starch (Glu)

i. Amylose is the linear polyglucoseii. Amylopectin is the branched polyglucose

4. Carbohydrate digestion starts in the mouth and continues in the intestinea. Salivary α –Amylase is inactivated by low pH of stomach (stomach doesn’t

digest carbohydrates)i. The end products of starch digestion by α –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

Name Origin Acts onα -Amylase Salivary glands Glu-α(1à4)-glu

in starchα-Amylase Pancreas Glu-α(1à4)-glu

in starchMaltase Intestinal mucosa Glu-α(1à4)-glu

in maltoseIsomaltose Intestinal mucosa Glu-α(1à6)-glu

in isomaltose and branched dextrins

Sucrase Intestinal mucosa Glu-α(1à2) β-fru in sucrose

Lactase Intestinal mucosa Gal- β(1à4)glu in lactose

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 à glucose and galactose

c. Intolerance : lactose passes further down GI tract and its metabolism by bacteria in colon à 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-β(1à4)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; arabinose7. 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. Does not require metabolic energyc. Requires a concentration gradient maintained by rapid removal of the transported sugar from inside

the cell to the capillaries

d. E.g. fructose; mannose; also glucose absorption into intestinal cells (in addition to active transport in intestine) à glucose release is facilitated transport, first stop is the liver

8. Active transport a. For glucose & galactose in intestineb. Allows absorption against a concentration gradientc. 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

Lumen Cell Blood Other cells[glucose] Medium High Medium Low

Transport Active àCell FacilitatedàBlood

FacilitatedàCell

9. Transportersa. 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 insulin1. GluT 2: liver, pancreas, intestine

10. Liver, RBC, and brain cells : carrier-mediated transport is not stimulated by insulin11. Muscle, fat, and WBC : glucose absorption is stimulated by insulin which acts by

mobilizing the transporters from stores on the membranes of the ERa. Insulin reduces circulating

blood glucose by stimulating 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

12. Diabetics &Blood Glucose Levela. The rise in concentration reflects:

i. Absorption into the bloodii. 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 liverb. Doubling of glucose after a meal; return

within 2 hours

c. Diabetes: start higher, go higher, come down slower13. 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

15. Glucose is phosphorylated quickly when it is absorbed by isoenzymes (glucokinase or hexokinase)

a. G6P is charged and cannot exit the cell

Enzymes Distribution Km for glucose

Effect Regulation

Hexokinases All tissues <0.2 mM

Low Km (high affinity; essentially always on)

Feedback inhibition by

product (G6P)Glucokinase Liver,

pancreatic islet β-cells

10-20 mM

High Km; High Vmax à high capacity but low

affinity

Substrate concentration and

amount of enzyme; Most active when BGL is high

16. Hexokinase a. Can phosphorylate other hexoses (e.g. fructose)b. 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 onc. If cell has enough G6P, it doesn’t 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) & pancreasb. High Vmax allows liver to effectively remove excess glucose, and minimize

hyperglycemia after eating

c. High BGL àIncreases 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 (don’t want it to be product inhibited); want liver to be able to continue in high glucose conditions

e.

e. Acts as part of glucose sensor system in pancreatic islet β-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 BGLà high ATP à shuts off potassium channel à insulin release

1. High BGL 2. Activate glucokinase3. Beta cell make more ATP

a. Which regulates the ATP-K channel that maintains cell polarity

4. Depolarizes the cell5. Calcium influx6. Release of insulin

18. Diabetesa. Type I diabetic

i. Higher resting glucose concentrationii. Higher peaks in plasma glucose after meal

iii. Return to baseline sloweriv. Not producing insulin necessary to control blood sugar

b. Type II i. Have the pancreatic islet β-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 β-cells to not respond normally to ingested glucose, so they do not secrete insulin normally

3. Mild to moderate hyperglycemia4. 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. Glucose to Pyruvate (aerobic) or Lactate (anaerobic) yielding 2ATP/mole glucoseb. 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 + ADP3. Irreversible

ii. Phosphohexose isomerase1. Input: Glucose-6-Phosphate2. Product: Fructose-6-Phosphate

iii. Phosphofructokinase 1 (PFK 1)1. Input: Fructose-6-Phosphate + ATP2. Output: Fructose-1,6-bisP

3. Inhibited by excess ATP, PEP, Citrate, glucagona. 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, Pia. 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

c. Dephosphorylation of PFK-2 drives glycolysis5. *First step committed uniquely to glycolysis 6. PFK1 deficiency : exercise intolerance in skeletal muscle

iv. Aldolase 1. Input: Fructose-1,6-bisP2. Product: two triose-phosphate units

a. Glyceraldehyde-3-P à substrate for next step of glycolysisb. Dihydroxyacetone-P

3. Possible next step: Triose phosphate isomerase (TIM)a. Input: dihydroxyacetone-P b. Product: glyceraldehyde-3-P

c. Pathway only favored when product is removedv. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH)

1. Input: glyceraldehyde-3-P + NAD+ + Pi2. Process: oxidation

a. Electrons are accepted by NAD+ coenzymeb. Reaction requires Pi for formation of high energy phosphate bond (at C1) which allows

transfer of the phosphate to ADP3. Product: 1,3-bisphosphoglycerate + NADH + H +

4. Enzyme displays negative cooperativity

a. Binds 4 molecules NAD+ but it becomes harder to add each successive moleculeb. 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 doesn’t get saturated immediately

vi. Phosphoglycerate Kinase (PGK)1. Input: 1,2-bisphosphoglyerate + ADP2. Product: 3-phosphoglycerate + ATP

vii. Phosphoglycerate Mutase (PGM)1. Input: 3-phosphoglycerate2. Product: 2-phosphoglycerate

viii. Enolase 1. Input: 2-phosphoglycerate2. Process: removal of water with creation of high energy phosphate bond

a. Strongly inhibited by fluoride3. Product: Phosphoenolpyruvate

ix. Pyruvate Kinase 1. Input: Phosphoenolpyruvate + ADP

2. Irreversible reaction 3. Stimulated by fructose-1,6-bisphosphate (feed forward stimulation)

4. Inhibited by: alanine, NADH, ATP, fatty acids, and succinyl-CoA5. Product: Pyruvate + ATP

6. PK deficiency causes hemolytic anemiax. Next step happens under anaerobic conditions: Lactate dehydrogenase

1. Input: Pyruvate + NADH + H+

2. Product: Lactate + NAD +

3. This step is required for regeneration of NAD+

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

3. Poisons can affect glycolysisa. 2-F-Deoxyglucose at hexokinase

i. Allows you to use PET to detect places where a lot of glycolysis is going on à tumors

b. Arsenate affects G3PDH i. 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

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 D’s”)

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

6. Irreversible steps are sites of regulation and are inhibited under resting conditionsa. 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-2,6-bisphosphate)

1. F-2,6-BP is made from F6P by PFK-IIii. 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 à activated

PFK2 makes more F2,6BP to positively regulate PFKI to go through the cycle and increase glycolysis

7. Connecting glycolysis with metabolism of pentoses, lipids and amino acids

Allosteric Regulation Phosphorylation Regulation

Induced Enzyme

RegulationRegulated Enzyme Activator Inhibitor Functional State Induced

byHexokinase Glucose-6-P

Phosphofructokinase I Fructose-2,6-bisP

ADP, AMP

Pi

ATPCitrate

Fatty AcidsPEP

GlucagonPyruvate Kinase Fructose-

1,6-bisP (fructose-2,6-bisP)

ATP, NADHAlanine

Fatty acidsSuccinylCoA

Inactive when phosphorylated

High carb & insulin

Glucokinase High BGL

High carb & insulin

Intermediate Can be used for:Glucose-6-phosphate Glycogen, polysaccharides, glycoproteins,

pentoses, NADPHFructose-6-phosphate &

glyceradldehyde-3-phosphatePentoses

Dihydroxyacetone-phosphate Glycerol phosphate à phosphatidic acid à neural fat & phospholipids

3-phosphoglycerate Serine1,3-bisphosphoglycerate 2,3-bisphosphoglycerate (BPG)

Pyruvate Amino Acids (Alanine)Acetyl CoA à fatty acids, cholesterol,

steroid hormones

8. Tissue-Specific DifferencesCell/ Tissue Major ATP Source

Erythrocytes Glycolysis onlyHeart muscle, Brain Oxidative metabolism, under all conditions

Skeletal muscle 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 defectsb. Skeletal muscle

i. Pyruvate formed enters mitochondria for oxidative metabolismii. 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 muscleiv. Lactate is transported to the liver for resynthesis

c. Cardiac muscle i. Lactate dehydrogenase is inhibited as pyruvate concentration

increases à 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 RBCEnzyme Stimulating Regulator (indicative of low energy state)

Hexokinase Low G6PPhosphofructokinase I Low ATP

High AMPPyruvate kinase High F-1,6-BP

16. OTHER SUGARS (FRUCTOSE, MANNOSE & GALACTOSE) & ALCOHOL

a. Sorbitol: can interconvert fructose & glucosei. Aldol reductase

1. Input: glucose + NADPH2. Product: Sorbitol + NADP

ii. Sorbitol dehydrogenase 1. Input: sorbitol + NADP

2. Product: fructose + NADPH

b. Fructose:i. Fructokinase

1. Input: fructose + ATP2. 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 urinec. 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 trioses1. One is phosphorylated (dihydroxyacetone-phosphate)

a. Triosephosphate isomerse : DHAP à G3P à glycolysis2. Glyceraldehyde kinase : glyceraldehyde à G3P à glycolysis3. 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 à 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-phosphatei. Accumulation is associated with depletion of ATP and Pi

ii. Changes in blood concentrations of metabolites1. Fall in glucose: F1P inhibits release of glucose from glycogen2. Fall in free phosphorus: Pi is used in re-synthesis of ATP; phosphorus

becomes trapped in F1P3. Rise in uric acid : from increased synthesis & breakdown of purine

nucleotides

d. Mannose:i. Hexokinase

1. Input: Mannose2. Product: Mannose-6-phosphate

ii. Phosphomannose isomerase 1. Input: Mannose-6-phosphate2. Product: fructose-6-phosphate à glycolysis

e. Galactose: (lactose = glucose + galactose)i. (1 st ) Galactokinase

1. Input: galactose2. Product: galactose-1-phosphate

a. Cell must make Uridine diphosphoglucose to use this3. Galactokinase Deficiency

1. High levels of blood galactose2. Some is reduced to galactitol (via aldol reductase) which can

lead to development of cataractsii. UDP-glucose pyrophosphorylase

1. Input: glucose-1-phosphate + uridine triphosphate (UTP) + Pi2. Product: uridine diphosphoglucose (UDP-glucose)

iii. (2 nd ) Galactose-1-phosphate uridyltransferase (UDP-Glc) a. Input: UDP-glucose + galactose-1-phosphateb. Product: UDP-galactose + glucose-1-phosphate

iv. Galactose-1-phosphate uridyltransferase deficiency

1. Classic Galactosemia ; Autosomal recessive2. Infant: cannot utilize galactose; must exclude from diet

a. galactose-1-phosphate accumulates; some is made into galactitol-1-phosphate à 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 : Gal actose 1 P hosphate U ridyl T ransferase .4. UDP-galactose-4-epimerase

a. Input: UDP-galactose + NAD+

b. Product: UDP-glucose + NADHc. 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. (3 rd ) Phosphoglucomutase 1. Input: glucose-1-phosphate2. Process: isomerization (~phosphoglycerate mutase reaction of

glycolysis)3. Product: glucose-6-phosphate à glycolysis

1. Alcohol Metabolismf. Oxidation of ethanol to acetaldehyde:

three different pathways:i. Cytosol

1. Alcohol dehydrogenase (ADH) &

NAD+

2. ADH has broad specificity3. There are 5 different ADH genes whose polymorphisms contribute to the ability of people to

oxidize ethyl alcoholii. Smooth ER

1. Microsomal ethanol oxidizing system (MEOS)2. Becomes important in high alcohol intake3. Alcohol can also interfere with cytochrome P450

iii. Peroxisomes1. Catalase

g. Acetaldehydei. Reacts with amino groups & proteins (cross-linking)

ii. Inhibits mitochondrial functions 1. Feedback loop: high acetaldehyde impairs mito function

leading to accumulation of more acetaldehydeiii. 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 formsa. Wild type: active enzymeb. Second form: inactive

i. Dominant allele; reduced ability to oxidize acetaldehyde à vasodilation, facial flushing

iv. AcetylCoA Synthetase 1. Input: acetate + ATP + CoA2. Product: acetylCoA + AMP + PPi3. Allows recovery of small amount of energy

h. Acidosisi. Excess acetate appears in the blood

ii. Deficiency of NAD+ leads to increased conversion of pyruvate to lactate by lactate dehydrogenase

i. Gluconeogenesisi. 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 liverk. Vitamin deficiency

i. Pyridoxine or folate deficiencies à 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. Poisonsi. Methanol (Moonshine)

1. Metabolized by same enzymes that metabolize ethanol2. Alcohol dehydrogenase activity on it produces formaldehyde and formic acid à

death3. Supplementing with ethanol can reverse condition

ii. Antifreeze1. Ethylene glycol is metabolized to glycoaldehyde by alcohol dehydrogenase

a. Then to glycolic acid by aldehyde dehydrogenase

b. Results in severe acidosis

17. OXIDATIVE METABOLISM - THE TRICARBOXYLIC ACID CYCLE

1. TCA: Mitochondria Matrixa. Inner membrane contains a carrier protein that allows pyruvate to enter the

matrix2. Citrate is exported from the mitochondria to allow fat synthesis & inhibit glycolysis

a. Acetyl-CoA is converted to citratei. There is no way to get acetyl-CoA directly out of mitochondria; must

convert to citrate first3. CAN cross mitochondrial membrane

a. Pyruvate; citrate; isocitrate; α-ketoglutarate; succinate; fumarate; malate; ATP, ADP, Pi

4. CAN NOT cross mitochondrial membrane a. Acetyl CoA; Oxaloacetate; NAD+, NADH

5. Pyruvate Dehydrogenase a. Substrate: Pyruvate (3C) + CoA + NAD+

b. Product: Acetyl(2C)-CoA + NADH + H+ + CO2

c. Regulation:i. Not reversible

ii. Inhibited by1. Acetyl CoA; NADH, ATP

a. Activates the kinase which phosphorylates pyruvate dehydrogenase into its INACTIVE form

b. Kinase: Phosphorylated = ACTIVE à PDH INACTIVE

c. Kinase Inhibited by products (pyruvate, NAD+, CoASH); dichloroacetate

iii. Activated by1. Insulin, Mg2+, Ca2+, pyruvate, NAD+, CoASH,

Dichloroacetatea. Phosphatase activated by Ca2

+ (in exercising muscle) & insulin (well-fed state) à means you want to do a lot of glycolysis & TCA cycle

b. Phosphatase: Dephosphosphorylated = ACTIVE à PDH ACTIVE

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

3. Deficiency leads to confusion, irritability, weight loss, edema, heart failure: Wernicke’s Encepalopathy (Wernicke-Korsakoff), beri-beri (wet or dry)

ii. Lipoate : active arm on E21. Transfers acetyl group to CoA, and electrons to a coenzyme (FAD) on E3

2. Target for Trivalent arsenic (AsO2-) (arsenic poisoning)iii. FAD : Flavin Adenine Dinucleotide: Coenzyme on E3

1. Derived from Vitamin B2 (riboflavin) iv. Coenzyme A/ Acetyl Coenzyme Av. NAD+

e. Leigh’s Disease i. Deficiency in PDHii. Degeneration of CNS; Neurological affects: loss of head control, motor skills, seizures;

Lactic acidosis can lead to impairment of respiratory & kidney functioniii. 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 à citrate

ii. Irreversible; Regulatediii. Inhibited by: NADH; succinyl CoAiv. Rate increases as [OAA] & [Acetyl CoA] increase

b. Aconitase i. Citrate à isocitrateii. 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+ à α-ketoglutarate + NADH + H+ + CO2

d. α-ketoglutarate dehydrogenase i. α-ketoglutarate + NAD+ à Succinyl-CoA + NADH + H+

ii. Five coenzymes: TPP, lipoate, CoASH, FAH, NAD+

1. ~pyruvate dehydrogenaseiii. Inhibited by: NADH; ATP; succinyl CoAiv. Lipoate is the target of arsenic

e. Succinyl CoA synthetase i. Succinyl-CoA + GDP + Pi à Succinate + GTPii. Succinate is symmetrical

f. Succinic acid dehydrogenase i. Succinate + FAD à Fumarate + FADH2

ii. Inhibited by: OAAiii. Activated by: ATP; Pi; succinate

g. Fumarase i. Fumarate à L-Malate

h. Malate dehydrogenase i. L-Malate + NAD+ à Oxaloacetate (OAA) + NADH

Regulated Enzyme Activator (‘low energy state’) Inhibitor(‘high energy state’)

Citrate synthase NADHATP

Succinyl CoA

Isocitrate dehydrogenase Isocitrate (insulin regulation results in citrate buildup)

ADP, AMPNAD+

Ca++

NADHATP

α-ketoglutarate dehydrogenase

Ca++ NADHSuccinyl CoA

7. Reduced coenzymes come from:a. NADH : Pyruvate dehydrogenase; Isocitrate dehydrogenase; α-ketoglutarate

dehydrogenase; Malate dehydrogenaseb. FADH2 : Succinate dehydrogenase

8. GTP comes from: Succinyl-CoA synthase9. Anaplerotic pathways

a. Need to replenish the intermediatesi. Glutamate dehydrogenase: à α-ketoglutarate

ii. Malic enzyme: Pyruvate à Malateiii. PEP carboxykinase: PEP à OAAiv. Pyruvate carboxylase: Pyruvate à OAAv. Transminase: Amino acids à OAA

10. TCA Cycle Intermediate linksa. All intermediates except malate, isocitrate, and citrate

(M.I.C.) can be turned into amino acidsb. Acetyl CoA à fatty acids & steroid biosynthesisc. Citrate à fatty acids/ sterol synthesis (cytoplasm)d. α-ketoglutarate à glutamate à amino acid purinese. Succinyl-CoA à hemef. Malate à glucose synthesis (cytoplasm)g. Fumarate urea cycle (tyrosine & phenylalanine breakdown)

h. Oxaloacetate ài. OAA formed by pyruvate carboxylase

pyruvateii. à asparate à amino acid purines/ pyrimidines

iii. à Interconverted with malate & exported to cytoplasm to act as substrates for gluconeogenesis

1. Convert to malate à generate NADH in cytoplasm2. Or can convert to asparate

18. THE PENTOSE PHOSPHATE PATHWAY

1. Using G6P for the biosynthesis of pentoses or formation of NADPHa. 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+ à 6-phosphoglucono-δ-lactone + NADPH + H+

2. Committed step; rate limiting3. Coenzyme: NADP+

4. Activated by: Insulin5. Inhibited: Allosterically by NADPH (potent competitive inhibitor)

a. Has a lower Ki than Km of NADP+ when present at higher concentrations

b. As NADPH is depleted more NADP+ is formed

c. High NADP+ stimulates Glucose 6-P dehydrogenase and NADPH production

6. G6PDH deficiency a. Precipitation of hemoglobin occurs due to disulfide

bond formation between Hb moleculesb. Extremely common; May present with: Dark colored urine; Low RBC

count; RBC with inclusion bodies; Elevated reticulocyte count; Low

hemoglobin; Elevated serum bilirubin

c. Clinical disease is related to degree of defecti. Class I: <2% enzyme activity à very severe

ii. Class III (A-): 10-50% enzyme activity à modest1. Patient with 10% of normal activity have enough to

generate NADPH under normal conditions.ii. Lactonase

1. 6-phosphoglucono-δ-lactone + H2Oà 6-phosphogluconate

iii. 6-phosphogluconate dehydrogenase 1. 6-phosphogluconate + NADP+ à Ribulose-5-P + NADPH2. 2nd formation of NADPH molecule

b. Isomerization & Epimerization i. Ribulose-5-Isomerase (“phosphopentose isomerase”)

1. Ribulose-5-P à Ribose-5-Pii. Ribulose-5-Epimerase (“phosphopentose epimerase”)

1. Ribulose-5-P à Xylulose-5-P

3. Transketolase a. Xylulose-5-P à Ribose-5-Pb. Transfers 2 Carbon Units Using

Thiamine (TPP)c. Wernicke-Korsakoff Syndrome

4. Transaldolase a. Transfers 3 carbon units

5. Four Modes of the PP Pathway:a. Balanced need for NADPH and R5P b. Need R5P (more than NADPH)

i. Rapidly dividing cellsc. Much more NADPH than R5P is needed

i. Synthesis of fatty acids in adipose tissued. Both NADPH and ATP are required; R5P can be converted to pyruvate which can be oxidized to

generate more ATPi. Biosynthesis

6. Net result: Oxidation of G6P, a 6 carbon sugar, into a 5 carbon sugari. 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-6-phosphate 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 & nucleusi. Gets reduced to NADH

ii. Glycolysis oxidation reactions (usually have with dehydrogenase enzyme reactions)

iii. Want cell to have high NAD and low NADHb. NAPH : coenzyme of reduction in cytosol

i. Gets oxidized to NADPii. Making fat (unsaturated to saturated double bone)

iii. Want high NADPH, low NADP (would drive reactions the opposite way)

8. Oxidative Damagea. superoxide dismutase converts superoxide to hydrogen peroxideb. 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

d. Defect means you cant detoxify peroxide and damages RBCsi. 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 mealb. Gluconeogenesis

i. Sustains BGL for many days in absence of carbohydrate intake2. Kidneys produce 10-20%, but more on fasting

3. Use 2 pyruvate, 4 ATP, 2 GTP, 2 NADH à get ONE molecule of glucose4. 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 à inhibition of earlier enzymes don’t affect as severely

5. Gluconeogenesis goes through multiple (three different) cellular compartmentsa. Mitochondria à cytoplasm à ERb. 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 mitochondriai. Conversion to PEP (through the action of the mitochondrial PEPCK)

ii. Transamination to aspartateiii. 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

a. Pyruvate Carboxylase i. Pyruvate + ATP + CO2-Biotin-Enzyme à OAA ii. Carboxylases add HCO3

- (i.e. CO2) to substrate

iii. Coenzyme: Biotin1. 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 à P-enolpyruvate (PEP) + CO2 + GTP

ii. Cofactor: GTPiii. Can occur in either cytosol or mitochondriaiv. Regulated by glucocorticoids such as cortisol

c. Fructose-1,6-Bisphosphatase i. F-1,6-BP à F6P

ii. Activated by: Citrateiii. Inhibited by: AMP; F-2,6-BP (low energy levels)iv. *Regulation is the opposite of PFK1

d. Glucose-6-Phosphatase

e. Regulation

i. PFK2: enzyme that synthesized F2,6BP which is a potent activator of glycolysis (PFK1) & inhibitor of gluconeogensis (FBPase)

ii. Insulin promotes the dephosphorylationà Stimulates phosphataseiii. Glucagon stimulates cyclic AMP & phosphorylation of PFK2 àF2,6BP hydrolyzed to

F6P1. Glycolysis decreases/ Gluconeogenesis increases (Fbase not inhibited)

7. Glucagon a. Liver: Stimulates gluconeogenesis and glycogenolysisb. No effect on muscle (no receptor)

8. Insulin a. Muscle: stimulates glucose uptakeb. Liver: Stimulates glycogenesis

Metabolic Actions of Insulin and Glucagon

Fatty acid uptake and release in fat.

InsulinStimulates synthesis of triglycerides (TG) from free fatty acids (FFA); inhibits

release of FFA from TG.

Glucagon Stimulates release of FFA from TG.

Liver glycogen

Insulin Increases synthesis and thereby glucose uptake and storage.

Glucagon Stimulates glycogenolysis and glucose release.

Liver gluconeogenesis

Insulin Inhibits, saves amino acids.

Glucagon Stimulates, glucose synthesized and released.

Glucose uptake, skeletal muscle

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

Glucagon 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.

9. Cori Cycle a. RBC or Muscle: Glucose àLactate 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 deali. Get 2 ATP in muscle and costs 6 ATP

in liver

10. Cahill Cycle a. Muscle: Conversion of pyruvate to alanineb. Converted back to pyruvate and then to glucose

in liver11. Oxidizing alcohol generates excess NADH, depriving liver of gluconeogenic

substratesa. Production of NADH by alcohol metabolism blocks gluconeogenesis by

converting pyruvate back to OAA rather than lactate; also blocks conversion of malate

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-6-phosphatase)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 BGLa. 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 (McArdle’s Disease).

ii. Product is glucose-1-phosphateb. 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 glucosed. Glycogen phosphorylase two distinct conformational states: a T (for tense,

less active) and R (for relaxed, more active) state; i. Low BGL à glucagon à Phosphorylate Glycogen

phosphorylase-a (R/ active state) à MORE ACTIVE à Breakdown Glycogen

1. Also enhanced by binding of AMP2. 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 4’OH of a glucose at the end of the chaini. Requires a primer which can be made by glycogenin

ii. This is the regulated stepb. 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 à Insulin à activate protein phosphatate à Dephosphorylate Glycogen synthase à MORE ACTIVE à Synthesize Glycogen

ii. Low BGL à glucagon à activate protein kinase à Phosphorylate Glycogen synthase à Less active

10. Glycogen Storage Diseases

Enzyme Location Glycogen Features Presentation Treatment

Type IVon

Gierke

Glucose-6-Phosphatase LiverKidney

Increased amountNormal structure

Severe hypoglycemiaHepatomegaly

Lactic acidosisHypoglycemiaHyperuricemiaHyperlipidemia

Constant Glucose infusion

Type IIPompe

α-1,4 glucosidase (lysosomal)

Everywhere Large increaseNormal structure

Cardiorespiratory Failure

HepatomegalyHypotonia Muscular weakness

Enzyme replacement

therapy

Type IIICori

Debranching Enzyme MuscleLiver

Increased amount

Similar to but milder than Type I Heptomegaly

High CarbHigh Protein

Short outer

branches

(enlarged liver)Hypoglycemia, Hyperlipidemia

Short statureType IVAndersons

Branching Enzyme Liver Normal amountLong outer

branches

HepatomegalyFatal

Type VMcArdle

Phosphorylase Muscle Increased amountNormal structure

Exercise problemsCramps

Burgundy colored urine

Type VIHers

Phosphorylase Liver Increased amountNormal structure

Similar to but milder than Type I

Hypoglycemia, Hepatomegaly Lactic acidosis

Carb Supplements

Type VIITauri’s

Phosphofructokinase1 Muscle Increased amountNormal structure

Like Type VExercise

intolerance

Muscle cramps Blood lactate not elevated hemolytic

anemia

Andersons is only disease of glycogen synthesis

Glycogen storage: names of types I through VI"V iagra P ills C ause A M ajor H ardon ":

Von Gierke'sPompe's

Cori'sAnderson'sMcArdle'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 à G6P cant convert to free glucose to be releasedb. Rate of glycolysis

i. High rate of glycolysis due to high [G1P] from glycogen breakdown

c. Rate of glycogen synthesisi. Decreased

d. Source of free blood glucosei. Break down glycogenà

1. Release free glucose2. Rest of G1P (10:1) goes through glycolysis

e. Rate of pentose synthesisi. Increase [G6P] à Increase PPS

ii. Breaking down nucleotides à increase uric acid in blood1. High level of glycolysis & lactic acid

f. Accumulation of lipids in liver and bloodi. NADPH & substrates (DHAP, citrate, acetyl-CoA, G6P) are still

available for FA synthesisg. Development of gout and approaches to control of the consequences of the

diseasei. Constant supply of glucose