BIOCHEMISTRY

28. Protein Digestion

1. Amino acids in excess of requirements are Sources and Uses of Amino Acids degraded to products that are either oxidized Body Protein for energy or converted into carbohydrates Dietary Protein 220 g/d Synthesis of 70-100g/d or fats Nitrogencontaining 2. Amino acids are maintained at certain levels Digestive Enzymes Compounds 70-100g/d in the blood for use by the body Amino Acid Pool a. Amino acid metabolism = Feces NITROGEN metabolism 10g/d Degradation for energy b. These amino acids come from two or to make glucose Protein Synthesis sources: our own body breakdown In Balance (digestive enzymes+body protein): about 300 g/d (about ) and the digestion of dietary protein (about ) The body maintains a pool of amino acids in the blood stream and tissues for c. Major amino acids in blood: protein synthesis, to use as fuel, or for conversion into other substances. The ONLY significant source of nitrogen in our diet is through protein Alanine and Glutamine 3. Dietary protein cannot be absorbed intact and is first hydrolyzed to its constituent amino acids (stomach small intestine liver (via portal vein)) 4. Three stages of protein digestion: a. Gastric (pH 1.5) i. Glands of the stomach lining secrete acid and pepsinogen ii. Acid denatures proteins; pepsin produces peptides 1. Pepsinogen is not active at pH > 2 due to a pro-peptide (prosegment) at the active site; autoactivation at pH < 2 results in active pepsin iii. Peptides enter the small intestine b. Intestinal (pH 6-8) i. Acid chime enters duodenum where proteolysis occurs ii. Partially digested protein triggers the release of hormones (secretin and cholecystokin) from specialized mucosal endocrine cells iii. Results in contraction of gallbladder and release of an alkaline secretion from pancrease containing bicarbonate and a mixture of endopeptidases (trypsin, chymotrypsin, and elastase; a.k.a serine proteases) and the exopeptidases (carboxypeptidase A and B)
1. 2. Endopeptidases: hydrolyze internal peptide bonds Exopeptidases: snip off N or C terminal residues

iv. Enzymes in the duodenum digest long peptides into short ones v. Enzymes of intestinal lumen (aminopeptidases and dipeptidases) produce most amino acid vi. Enzymes inside intestinal cells hydrolyze to amino acids blood c. Pancreatic i. Pancreas secretions initially produce inactive proteases (bicarbonate & zymogens) 1

1. Trypsinogen (from pancreas) (via enteropeptidase) trypsin 2. Enzyme enteropeptidase (secreted from small intestine) is the key event in activation of trypsinogen; conformation change a. Secreted from the luminal surface of the small intestine under the influence of CCK b. Newly formed trypsin acts autocatalytically by hydrolyzing the same bond as enteropeptidase c. Trypsin activates other zymogens: i. Chymotrypsinogen; Proelastase; Procarboxypeptidase ii. Pancreatitis: tissue damage and pain due to uninhibited trypsin 1. Non-hereditary: Zymogens dont get properly released due to pancreatic duct blockage; get activated and overwhelm trypsin inhibitors a. Trypsin inhibitor-trypsin complex is so tight trypsin cant digest its peptide inhibitor 2. Hereditary: Change of a particular amino acid in the trypsin molecule can disrupt the interaction with its peptide inhibitor (a backup safeguard mechanism in the pancreas) 3. Acute: Pain is centered in the upper middle or upper left part of the abdomen. Often temporary 4. Chronic: diminished pancreas function 5. Amino acid absorption: uptake into and out of intestinal cells occurs through transporters

Enters portal vein on the way to the liver

a. Amino acid concentration low: lumen and blood b. Amino acid concentration high: inside intestinal cells i. Active transport using Na+ linked systems specific for groups of amino acids 1. There are at least 7 different brush border specific transport systems 2. E.g. A-system: small neutral amino acids; cysteine transporter ii. A gradient of sodium (outside high, inside low) is created by Na+/K+ ATPase iii. Transporters in the brush-border membrane simultaneously move Na+ and an amino acid across the membrane, using the energy of the Na+ gradient to concentrate amino acids within the cell

(low aa conc)

(higher aa conc)

(lower aa conc)

6. The kidney normally reabsorbs amino acids for re-use a. Is nearly 100% efficient b. Defects result in high levels of certain amino acids in urine (aminoaciduria) c. Cystinuria i. Genetic disorder; carrier rate: 1/150 (1/10,000 have the disease) ii. Defective re-uptake membrane transporter in kidney glomeruli leads to high levels of cystine in urine is oxidized leading to kidney stones iii. Treatment involves making the urine more alkaline d. Hartnup disease i. Defects in a different transporter, results in the abnormal excretion of several neutral amino acids, including tryptophan into the urine; also deficient in absorption in the intestine ii. Tryptophan can be converted into niacin (and also Serotonin and Melatonin) 1. Niacin is a precursor to Nicotinamide (necessary component of NAD+) iii. Genetic disease with a pellagra-like presentation (dermatitis, diarrhea and dementia although intermittent and slightly less severe in comparison) due to tryptophan deficiency, especially after periods of poor nutrition
iv. The effects of the disease occur mainly in the brain & skin, even though the underlying disease is in the cells of the kidney and intestine; Rash develops on parts of the body exposed to the sun; Mental retardation, short stature, headaches, unsteady gait, and collapsing or fainting are common; Psychiatric problems may also result such as anxiety, rapid mood changes, delusions, and hallucinations)

v. E.g. Niacin requirements study of 1952: Seven subjects were maintained on diets low in niacin and tryptophan for from 40 to 135 days and the urinary excretion of niacin metabolites was determined. Clinical signs of pellagra developed in the three subjects who remained on one of these diets for more than 50 days. 7. Intracellular protein breakdown occurs in response to stresses (rev up the machinery needed for muscle breakdown; muscle contributes more than 60% of the total amino acid pool of blood) 8. Mechanisms of protein degradation 9. Lysosomal system (minor contribution) a. Cathepsins (lysosomal proteases) are effective for bacteria, apoptotic fragments, extracellular proteins (those in LDL), etc b. Inhibitors of lysosomal function (e.g. chloroquine) have little effect on the degradation of most intracellular proteins 10. Digestive enzyme turnover
Cachexia is the loss of weight, muscle atrophy, fatigue, weakness and appetite in someone who is not actively trying to lose weight.

a. The digestive enzymes are themselves being degraded and contribute amino acids back to the pool (~70 grams/day) i. About the same level of digestive enzymes are being synthesized 11. Ubiquitin/ Proteasome Pathway (UPP): the major pathway for the selective and coordinated degeneration of intracellular proteins (e.g. muscle) a. Proteins destined for degradation are conjugated with multiple ubiquitins (marker polypeptides) i. One E1 enzyme, several E2 enzymes, and many specific E3 enzymes exist to add ubiquitin (Ub) to the 'target' proteina protein with a particular signal b. Final product is a Proteasome Functions polyubiquitinated protein destined for the proteasome in the cytoplasm 5 & 6: other non-Ub methods of c. Proteasome components sending proteins to the Proteasome i. Regulatory protein: selects substrates; removed Ub for Step 8 >> Step 9 recycling; edits wrongly tagged proteins Important point: Ubiquitin is ii. Barrel core: inside, recycled in a reaction by the Tap: Transporter Associated deubiquitinase (DUB) enzyme peptides are digested via with Antigen Processing proteases to peptides of 7 to 10 amino acids Abundant iii. Some of these peptides aminopeptidases inside most cells can be transported through the ER for presentation to the immune system by MHC Class I molecules; others are degraded to amino acids by other cytosolic proteases and aminoepeptidases
d. Regulation: i. Inadequate caloric intake: muscle proteins broken down to provide amino acids for gluconeogenesis, protein synthesis, and energy production; cellular content of specific E3 enzymes varies among tissues and physiologic states

e. Protein degrade at different rates; in general, proteins should be around for a long time (cell cycle regulated enzymes arent) i. Lactate dehydrogenase (t1/2 = 171 hours) ii. d-Aminolevulinate synthase (t1/2 = 1) iii. HMG-CoA reductase (t1/2 = 3) iv. p53 (t1/2 = 0.5) v. Histones (t1/2 = 2800) f. Ubiquitination Signals i. In the protein sequence; not variable 1. N-end rule (methionine is slowest) 2. PEST sequences 3. Destruction boxes ii. External factors; more variable 1. Phosphorylation 4

2. Denaturation/ damage 3. Facilitators/ Chaperones; interaction with other proteins determines the likelihood that a particular protein will be ubiquinated (e.g. HPV16 E6) HPV E6 facilitates the degradation of p53 4. E6 protein of HPV binds Form complex with target protein to the tumor suppressor p53 and to E6AP (a Increase (or decrease) likelihood of ubiquitination. particular E3 with a HECT domain); this interaction results in the ubiquitination and degradation of p53 and The presence of the viral E6 protein increases ubiquitination of p53 contributes to the by recruiting E6-AP, a E3 ubiquitin ligase oncogenicity of the virus p53, cell cycle, # of cells Certain types of HPV are associated with cervical cancer 12. Abnormalities in the UPP in disease a. Loss of function mutations resulting in increased stability of substrates i. E.g. cancer from stabilization of oncogenes b. Gain of function mutations resulting in decreased stability or half-life i. E.g. cancer from destabilization of tumor suppressors (p53) c. Parkinsons Disease Several mutations can cause Parkinsons Disease i. Most cases of Parkinsons disease are characterized by accumulation of ubiquitin conjugates (Lewy bodies = protein deposits) in the brain Parkin is a E3-type 1. The Lewy bodies cause a ubiquitin ligase is a loss of neuronal cells & loss deubiquitinase deubiquitinof dopamine, a neural ase transmitter 2. The most widely used form of treatment is L-dopa in Usually Lewy Bodies in various forms. surviving neurons ii. Several mutations can cause Loss of neuronal cells & loss of dopamine, a neural transmitter Parkinsons 1. Defect in Parkin protein: a ubiquitin-conjugating ligase 2. Malfunctioning deubiquitinase 13. Amino Acid Pools and Essential Amino Acids a. Free amino acid levels are low compared to that polymerized in proteins (about 0.5%); 95% is replaced every 10 minutes. b. Amino acids are transported in plasma (2-4 mM) to replenish intracellular amino acids (15-30 mM) c. Alanine and glutamine are the most abundant amino acids in plasma (it's alanine, glutamate, glutamine, and glycine inside cells)
UCH-L1

i. These do not reflect their abundance in proteins it's more their metabolism d. All 20 amino acids have to be maintained in the blood e. Some Amino Acids are Essential and must be obtained from degradation of ones own body protein or the diet i. Amino acids cannot be stored for later use; all 20 must be present in approximately correct proportions for protein synthesis ii. Adults should consume at least 55g protein per day, otherwise breakdown of body protein will occur f. Kwashiorkor results from inadequate protein or incomplete protein although total calories are ok i. Many children in West African villages show signs of kwashiorkor. Their stomachs are bloated [due to loss of plasma protein], their arms and legs are thin, and their skin is flaky. ii. Proteins lost in 'starvation' include digestive enzymes and albumin
Essential Amino Acids needed for "Complete Protein
Arginine Valine Phenylalanine Histidine Threonine Methionine Are valuable for his thriving metabolism Isoleucine Lysine Leucine Trptophan in lifes lunatic trip or PVT TIM HALL (using non-standard 1-letter abbreviations for the amino acids)
"These Ten Valuable Amino Acids Have Long Preserved Life In Man"

Comparing Protein Sources

ANIMAL 10 - 25% protein Easily digested Nearly complete AAs VEGETABLE 1 - 2% protein Incompletely digested Low in essential AAs
Lysine, methionine, threonine

(legumes & grains are complementary)

Thr Trp Val Arg His Lys Phe Leu Ile Met

Chemical Score is high Chemical Score is low

29. Protein Degradation & Nitrogen

Dietary Protein 70-100g/d

Body Protein 220 g/d

Digestive Juices 1. N-acetyl glutamate synthetase deficiency (NAGS) 70-100g/d Amino Acid Pool a. Tremors, slowness of motion, loss of balance, Feces stiffness 10g/d Degradation b. Treatment: Carbaglu pills Protein Synthesis 2. Nitrogen excretion in urine NH Carbon skeleton a. Ammonia (NH4+): normally < 0.1 mM in Urea glucose, fat, CO blood, a little in urine b. Urea: >90% of N in humans c. Uric acid: a little in urine; is a purine breakdown product; creatinine is proportional to muscle mass
3

Synthesis of Nitrogencontaining Compounds

Nitrogen Balance 3. Nitrogen of degraded amino acids shows up mostly as urea Balance a. Under conditions of constant weight (nitrogen Normal Adult, not gaining muscle or protein (weight stable) balance), only the urea level changes as a result of Dietary Protein Amino acid pool Urea changed protein intake b. No protein reserves created as a result of 'excessive' Body Protein protein intake Balance c. Only the carbons from excess amino acids liberated Positive Growth, Pregnancy, Muscle Building from proteins are stored (or burnt to CO2 as fuel) Dietary Protein Amino acid pool Urea 4. Nitrogen balance: urea levels are proportional to protein Body Protein (is increasing) load 5. Positive nitrogen balance: increased dietary protein; body protein is increased; the nitrogen is ending up in protein 6. Negative nitrogen balance: loss of protein due to the use of body protein 7. The carbons of excess amino acids are used; the nitrogen is waste a. Nitrogen is removed in three steps: i. Transfer to a common carrier (Glutamate) ii. Ammonia is re-generated in liver iii. Ammonia is incorporated into urea 8. Amino acids transfer their nitrogen via transamination reactions (readily reversible) a. Nitrogen of amino acids transferred to KG to turn into glutamate i. Major -keto acids: KG; OAA; pyruvate ii. Corresponding amino acids: glutamate; Ammonia is also produced from amides on asparate; alanine asparagine and glutamine iii. There are no keto-acids for lysine or threonine b. Enzyme: aminotransferase i. Coenzyme: PLP = pyridoxal phosphate Several reactions produce ammonia; what 1. Arises from Vitamin B6 do you do with the a. B6 deficiency associated ammonia? with dementia due to lack 3 ways to dispose of it: 1. Make glutamate of serotonin production 2. Make glutamine from tryptophan 3. Make urea 2. PLP exchanges a keto group for methylenyl amine group 9. The N content of Glutamate is used to make urea a. Most cells have plenty of transaminases and glutamate dehydrogenase b. Release of NH4+ in liver can be incorporated in urea c. Urea production is significant only in the liver (multiple enzymes are required)
(and digestive enzymes)

The reaction catalyzed by glutamate dehydrogenase is key

& ATP

& GDP

Glutamate Dehydrogenase

d. Glutamate dehydrogenase produces ammonia i. Reversible, regulated step ii. Forward reaction is fuel-burning iii. Reverse reaction can be used to fix ammonia iv. Located in mitochondria; there are abundant glutamate and KG, ATP/ADP transporters 10. Fixation of nitrogen is important for some tissues such as the brain, where ammonia is toxic; only the liver can package it into urea a. Glutamine Synthetase reaction can also fix ammonia in the brain, muscle, lungs, and adipose cells i. Glutamine is a major carrier of N in the blood; glutamine levels increase after a protein rich meal ii. Glutamine levels can only rise so much to absorb ammonia: when blood glutamine plateaus, blood ammonia is excessive (e.g. fatal for infant born into a family with a Arginase argininosuccinase history of hyperammonemia) 11. Urea Cycle
a. b. Some urea cycle enzymes increase 10-fold or more after a protein-rich meal The liver has ALL five enzymes & proteins for urea synthesis (requires other transporters, enzymes to function fully) 2 steps in mitochondria; 3 in cytosol i. Make NH4+ in the mitochondria of liver cells for incorporation into carbamoyl phosphate (and eventually, urea) ii. Ornithine enters mitochondria, combines with carbamoyl phosphate (catalyzed by ornithine transcarbamoylase), exits as citrulline iii. Production of carbamoyl phosphate is controlled by NAG, which is controlled by arginine iv. Urea is produced from arginine (catalyzed by arginase)
Argininosuccinate synthetase Ornithine transcarbamoylase (OTC)

Pi Stryer
2 ATP

c.

Carbamoyl-phosphate synthesis in the mitochondria is catalyzed by CPSI

NAG synthase is activated by arginine d. A deficiency in any of the enzymes results is treated with carbamoyl (a feed-forward disposal pathway) glutamate in elevated ammonia e. The availability of carbamoyl phosphate is key; adding arginine usually helps f. One nitrogen comes directly from NH comes from the glutamate dehydrogenase and the asparate, the other from NH4+ glutaminase reactions g. Ordinarily Careless Crappers Are Also Frivolous About The big picture of Nitrogen Excretion Urination (ornithine; carbamoyl phosphate; citrulline; aspartate; argininosuccinate; fumarate; arginine; urea) 12. Genetic Defects: Urea Cycle a. Features of an inoperative or stressed cycle: Developmental
+ 4

NAG is found only in mitochondria NAG synthase deficiency

The equivalent of several (4) ATP's is used to make 1 molecule of urea

delays; Mental retardation, seizure; Protein intolerance b. Diagnosis: Blood/urine amino acid analyses for ammonia (perhaps high arginine for arginase deficiency or high glutamine levels); Enzyme assay c. Treatment: Restrict protein (Nitrogen production); supplement with arginine; Increase Nitrogen excretion (using medications that bind amino acids: benzoate, or phenylacetate or phenylbutyrate) 13. The urea cycle interconnects with carbohydrate metabolism: OAA; KG, fumarate
The Urea cycle interconnects with carbohydrate metabolism

3 major points of connection: Aspartate/oxaloacetate Glutamate/a-ketoglurate Fumarate/malate

14. Arginine is a useful precursor for many compounds a. Needed for nitric oxide (NO) production with the production of citrulline (NO is a vasodilator needed for smooth muscle relaxation) b. Converted with ornithine via the urea cycle, which gets transaminated into something that turns into glutamate and prolineglutamate and proline c. Arginine is made from argininosuccinate and the action of argininosuccinase 15. Arginine, ornithine, citrulline, and proline are metabolically linked via a glutamate derivative

30. Amino Acid Breakdown

30. Amino Acid Breakdown the Carbon skeletons

Dietary Protein 70-100g/d Digestive Juices 70-100g/d Body Protein 220 g/d Synthesis (non-essential)

Fates of the amino acids:

Glycogenic or Ketogenic

Some amino acids have multiple entry points (Threonine, Tyrosine in 2 parts)

You can't get blood glucose from leucine and lysine

Amino Acid Pool

Conversion to purines, pyrimidines, heme, creatine, amines Degradation NH2 Carbon skeleton Carbo. Fat

Feces 10g/d Protein Synthesis

NH3 Urea

CO2

1. Amino acids released from proteins (mostly muscle) can be used for making glucose or for fuel 2. Glycogenic: after deamination, the carbon skeletons of the amino acids are intermediates of glycolysis or the TCA cycle and can contribute to the synthesis of glucose and glycogen via gluconeogenesis and glycogenesis a. Enter carbohydrate metabolism at one of five point: i. Pyruvate; -ketoglutarate; succinyl-CoA; fumarate; OAA b. Amino acids that have more than one point of entry: one part of each molecule contributes to acetyl-CoA, another part is converted to a gluconeogenic intermdiate 3. Ketogenic: amino acids that give rise to short chain fatty acids that undergo oxidation to ketone bodies or acetyl-CoA; these cannot be converted into carbohydrate because of the irreversibility of pyruvate dehydrogenase a. Lysine and leucine are metabolized to acetyl-CoA and hence don't make glucose Glycogenic Ketogenic Glycogenic & Ketogenic Alanine Glutamate Proline Leucine Isoleucine Arginine Glutamine Serine Lysine Phenylalanine Asparate Glycine Valine Threonine Asparagine Histidine Tryptophan Cysteine Methionine Tyrosine 4. Pyruvate pathway: Several glycogenic amino acids are metabolized to pyruvate ( alanine [tryptophan], serine[ glycine threonine], cysteine)
a. b. serine-pyruvate transaminase to make alanine and hydroxypyruvate in 2 more steps converted to glycerate-2-P and then to pyruvate The details of cysteine & threonine degradation (and tryptophan and histidine) are not examined in this course

c. Dehydratases acting on amino acids result in the production of different ketoacids i. The following are glycolytic substrates: 10

ii. Serine pyruvate glucose iii. Threonine -ketobutyrate glucose 5. -ketoglutarate pathway a. Proline, glutamine, arginine, glutamate are broken down to KG
2. a -Ketoglutarate Pathway
Arginine urea Ornithine a -Kg Glu Glutamate a -Ketoglutarate + NH 3 Glutamine
NH3

Arginine, Proline, Histidine, Glutamine, Glutamate

Glutamic g-semialdehyde Proline

NAD+ NADH

Histidine

3. 2. Succinyl CoA Pathway a -Ketoglutarate Methionine, Isoleucine, Valine Glutamate Pathway Threonine, Arginine, Proline, Histidine, Glutamine,
Arginine urea Methionine a -Kg Threonine Ornithine Glu

Isoleucine Glutamine
AT

Valine
NH3

Proline dehydrogenase AT

Proline

6. Succinyl-CoA Histidine a. Methionine, isoleucine, valine, threonine Propionyl CoA

3.AT Succinyl CoA Pathway - amino transferases
DH* - branched-chain ketoacid dehydrogenase (see section on maple syrup urine disease for Methionine more details)

a -keto-b-methylvalerate a -ketoisovalerate a -Ketoglutarate g-semialdehyde Glutamate Glutamic a -ketobutyrate DH* + NH 3 NAD+ DH* NADH DH* Proline NH3 pathway

Threonine, Methionine, Isoleucine, Valine Isoleucine Succinyl CoA AT Valine Succinate AT

Threonine

4. Fumarate Pathway Phenylalanine, Tyrosine a -ketobutyrate

NH3 Phenylalanine

a -keto-b-methylvalerate a -ketoisovalerate
DH*

Tyrosine

DH*

Fumarate

DH*

acetoacetate

AT - amino transferases DH* - branched-chain ketoacid C C dehydrogenase NH2 (see section on maple syrup urine disease for C HO C C C more details)

Propionyl CoA acetoacetate

fumarate

C C

Succinyl CoA COOH

Succinate

Note: a-ketobutyrate and other a-ketoacids are metabolized in reactions similar to pyruvate 5. Oxalacetate Pathway

4. Fumarate Pathway

Phenylalanine, Tyrosine

b. Transamination to form the corresponding -ketoacids AT acetoacetate Aspartate Oxaloacetate Asparagine acetoacetate c. Oxidative decarboxylation by a dehydrogenase complex: the branched chain C C NH 2 2-keto acid dehydrogenase (BCKADH), containing thiamine pyrophosphate, HO C C C C lipoate, and FAD H C C COOH fumarate i. Mechanism similar to pyruvate dehydrogenase ii. When BCKADH is missing, the 3 branched chain keto acids 5. Oxalacetate Pathway (isoleucine, valine, leucine) as well as -ketobutyrate (from NH3 AT methionine and threonine metobilism) accumulate Aspartate Oxaloacetate Asparagine 1. By reversal of the aminotransferase reaction, the blood levels of the branched chain amino acids also rise

dehydrogenase and require thiamine, lipoate, FAD Phenylalanine Tyrosine NH 3

Fumarate

11

2. Maple Syrup Urine Disease: individuals that lack the enzyme complex and build up keto acids, resulting in urine that smells of maple syrup a. Treatment includes severe restriction in the intake of branched chain amino acids iii. The propionyl-CoA derived from isoleucine, threonine, valine, and methionine are metabolized to succinyl-CoA 1. Requires biotin and vitamin B12 a. Vitamin B12 is required for metabolism of valine, isoleucine, & methionine
b. c. 3 to 40% of older adults have various degrees of B12 deficiency Symptoms range from lethargy and weight loss to dementia

2. Adenosylcobalamine is formed from Vitamin B12 a. adenosyl form of Vitamin B12 is needed for the mutase reaction b. The vitamin is absorbed using intrinsic factor, a glycoprotein in the cells lining the stomach (the actual absorption occurs in the small intestine) 3. Methylmalonic academia: failure to convert methylmalonylCoA to succinyl-CoA a. Several possible deficiencies: dietary intake of VitB12, lack of intrinsic factor, or an inability to convert to the adenosyl form, or deficient enzyme b. Results in hematopoietic and neurological disorders c. Treatment: administer large doses of Adenosylcobalamine 4. Mutase reaction defect; methylmalonate appears in the urine a. Underlying problem can be complex: missing enzyme or vitamin B12, problem with its absorption, or its processing b. Unresponsive to Adenosylcobalamine dosing 7. Oxaloacetate Pathway a. Asparagine is degraded to asparate (via asparaginase) which is then degraded to OAA (via asparate aminotransferase) i. The NH4+ of aspartate is passed on to -ketoglutarate to make glutamate in a transamination reaction 8. Fumarate pathway a. Phenylalanine tyrosine Dihydrobiopterin reductase fumarate & acetoacetate regenerates tetrahydrobiopterin b. The products are glucogenic & ketogenic c. Phenylalanine is an essential amino acid; when deficient, the *possible mutation metabolites made from it are *possible mutation also deficient d. Phenylketonuria (PKU) 12
(Tetrahydrobiopterin is synthesized from GTP)

Tyrosine becomes an essential aa in individuals with PKU

i. Build-up of phenylpyruvic acid; associated with mental retardation ii. Different forms; can be deficient in: 1. Phenylalanine hydroxylase in the liver involved in converting dietary phenylalanine to tyrosine 2. The ability to make tetrahydrobiopterin (a necessary cofactor)
a. 50% of PKU patients are responsive to tetrahydrobiopterin replacement iii. Guthrie test an assay for the presence of high levels of phenylalanine

iv. High concentrations of phenylalanine cause brain damage 1. High phenylalanine levels prevents other amino acids being transported into brain, perhaps also interferes with protein and neurotransmitter (serotonin) production
v. With phenylalanine metabolism blocked other minor compounds such as phenylpyruvate are produced 1. Urine is typically musty 2. Mothers with PKU must control phenylalanine levels in diet when pregnant

vi. Treatment: 1. Protein restricted diet, low phenylalanine 2. Supplement for Tyrosine, perhaps supplement a co-enzyme 3. Monitor serum levels of phenylalanine closely e. Alkaptonuria is characterized by dark urine i. Due to a deficiency in homogentisic acid oxidase ii. Later symptoms: arthritis, due to homogentisic forming crystals in the spine leading to cartilage damage and osteoarthritis 9. Inborn errors of amino acid metabolism are mainly pediatric diseases
a. b. Blood levels of the amino acid can increase to the point that the threshold for reabsorption by the kidney is exceeded and large amounts are excreted in the urine Because of common pathways for tubular reabsorption, an excess of one amino acid can also cause increased urinary excretion of other amino acids

Medical Condition Alkaptonuria

Defective Process Tyrosine degradation Urea synthesis Urea synthesis Urea synthesis

Defective Enzyme Homogentisate 1,2dioxygenase Arginase Argininosuccinate lyase Carbamoyl phosphate synthetase I Branched-chain keto acid dehydrogenase complex Methmalonyl-CoA mutase

Argininemia Argininosuccinic academia Carbamoyl phosphate synthetase I deficiency Maple Syrup urine disease (branchedchain ketoaciduria) Methylmalonic academia

Symptoms and effects Dark pigment in urine; latedeveloping arthritis Mental retardation Vomiting, convulsions Lethargy, convulsions, early death Vomiting, convulsions, mental retardation, early death Vomiting, convulsions, mental retardation, early 13

Isoleucine, leucine, and valine degradation Conversion of propionyl-CoA to succinyl-CoA

Phenylketonuria (PKU)

Conversion of phenyl-alanine to tyrosine

Phenylalanine hydroxylase

death Neonatal vomiting, mental retardation

31. Synthesis Using Amino Acids

1. Different organs use different amino acids differently a. Niacin b. Nitrous oxide c. Urea cycle makes arginine; glutamine from glutamate, and tyrosine from phenylalanine d. Synthesis of nitrogen containing compounds, including: i. Dopamine, thyroxine, melanin, glycine, norepinephrine, epinephrine, methionine, taurine, creatine/ creatinine 2. Compounds made from Tyrosine: a. Dopamine (a catecholamine): released by naturally rewarding experiences
[made in the brain]

i. Use of tetrahydrobiopterin in the hydroxylase reaction and the use of PLP in the decarboxylase reaction 14

b. Thyroxine: hormone that controls the rate of metabolic processes [made in the
thyroid gland]

i. Made from the iodination and covalent bonding of two tyrosine residues on thyroglobulin
ii. Thyroxine is cleaved from thyroglobulin and secreted from the thyroid

iii. Graves Disease: patients develop antibodies against a receptor that results in over-stimulated thyroglobulin 1. Too much thyroxine results in hyperthyroidism (fatigue, weight loss, increased appetite) c. Melanin: provides pigment in skin and eyes [made in the skin] i. Made from tyrosine via tyrosinase ii. Albinism: patients lack tyrosinase and are unable to make melanin, leading to sun sensitivity and improper eye development 3. Biosynthesis using Folate: Synthesis of Glycine, Serine, Cysteine & Methionine a. The synthesis of: Glycine & Serine & Cysteine (& Methionine) require 1carbon transfers Cofactor Group Transferred Biotin, PLP CO2 b. Several coenzymes are needed
c. Folates provide one-carbon fragments for amino acid & protein metabolism i. Are also involved in synthesis of purines & pyrimidines, nucleic acids, phospholipids, and regeneration of methyl methyl groups used for transamination by methionine
Tetrahydrofolate HC=O -CH2OH -CH3 S-adenosyl methionine -CH3

(formyl or forminino) (methylene) (methyl, for 1 reaction) (methyl, preferred)

ii. Low folates can block hematopoiesis, resulting in release of immature RBCs and megaloblastic anemia
d. Antifolates: folate metabolism is a target for antibacterial agents because of its central role in synthesis i. Bacteria can synthesize their own supply of folate, which can be blocked by sulfanilamides which do not interfere with folate metabolism in humans

ii. Methotrexate: structural analog of folate that interferes with rapidly growing cancer cells by interfering with the synthesis of pyrimidines e. Tetrahydrofolate (THF) is derived from folic acid
All but one of the glutamyl residues are removed in the intestinal mucosa, then free folic acid is reduced by dihydrofolate reductase to THF ii. THF is transported in plasma as the methyl derivative iii. Inside cells, THF is reconverted to the polyglutamyl forms (which are most effective for onecarbon transfers) i.

Most of the forms of folate can readily interconvert

Folate Dihydrofolate reductase

The CH3 form is the most abundant in blood plasma; it is formed in an irreversible reaction

iv. Methyl groups can be carried by N5 and N10 on the methylpterin ring v. By carrying methyl groups (in different formats) at the N5 and N10 atoms,

These forms of THF are known as the one-carbon pool

15

folates can carryout essential methylation reactions

vi. vii. Requirement for folates in nucleic acid synthesis is very important for rapidly growing tissues Folates are found in green leafy vegetables, beans, lentils, some fruits

viii. Spina bifida: results from low levels of folate during pregnancy 1. Associated with low blood levels of folate and vitamin B12, and high levels of homocysteine ix. 1-carbon transfers between serine and glycine use the methylene and tetrahydro forms of folate (via serine hydroxymethyl-transferase) 1. The tetrahydrofolate (H4 folate) is converted to the methylene form as glycine is made from serine (and vice versa)
x. 2. Serine is made from the glycolytic intermediate 3-phosphoglycerate One carbon fragments can also be derived from tryptophan and histidine

f. S-adenosyl methionine (SAM): can also transfer methyl groups in addition to THF i. Synthesized from methionine via methionine adenosyltransferase ii. Can be used in synthesis of epinephrine from norepinephrine (which is made from dopamine) iii. The production of creatine requires SAM 1. Synthesized from glycine, arginine, and methionine 2. Creatine phosphate is a high-energy compound that acts as a reserve energy supply in muscle by phosphorylating ADP to ATP (via creatine phosphokinase)
3. Creatine supplementation may also induce a cellular swelling in muscle cells, which in turn may affect carbohydrate and protein metabolism In summary, the predominance of research indicates that creatine supplementation represents a safe, effective, and legal method to enhance muscle size and strength responses to resistance training.

iv. Folate can be used to regenerate methionine from Sadenosylhomocysteine (SAH) 1. Methionine synthase uses methylTHF to methylate Vitamin B12 to make CH3-B12 (aka methylcobalamin cofactor) a. The enzyme uses CH3-B12 to remethylate homocysteine to methionine 2. Methylmalonyl mutase & methionine synthase are the only 2 enzymes known that require cofactors derived from Vitamin B12 a. The Folate Trap: The only way in which the THF in methyl-THF can be returned to the THF pool is via the B12-depedent methionine synthase reaction since the methylenetetrahydrofolate reductase (MTFR) reaction (leading to formation of methyl-THF) is essentially irreversible
b. Vitamin B12 deficiencies lead to: i. Defects in methionine synthase reactions ii. Build-up of methyl-THF iii. Decreased levels of available THF iv. A secondary deficiency in folate v. Deficiencies of functional folate can arise from a lack of dietary folate or vitamin B12

16

3. Methionine can also be regenerated from homocysteine using choline 4. Because homocysteine is used for other reactions, methionine can be in short supply g. Degradation of homocysteine
i. Homocysteine is constantly being produced in the body; some is being reconverted to methionine by methylation, some is degraded in a series of reactions that give rise to cysteine

ii. Cysteine Synthesis: two reactions, which are the only way to make cysteine 1. Cysteine is derived from methionine and serine 2. Cystathionine synthetase required PLP 3. If methionine is deficient, cysteine is also deficient 4. Degradation of methionine forms -ketobutyrate ( succinylCoA) 5. Taurine (2-aminoethanesulfonic acid) is derived from cysteine a. Is an important component of the substances found in bile (conjugated
with cholate) and can be found in the lower intestine

h. Homocytinuria: i. Usually due to a defect in cystathionine synthetase ii. Elevated blood methionine iii. Elevated urine homocystine (the oxidized product of homocysteine) iv. Accumulation and swelling of artery walls (plaque formation from excess homocysteine and LDLs); tall, thin stature (disturbance in bone development); vision problems; psychiatric disturbances; before the age of
30, almost one fourth of untreated patients die as a result of thrombotic complications

v. General treatment: restrict protein, supplement with vitamin-derived cofactors, supplementation with cysteine may help some symptoms vi. Supplementing with _______ can reduce homocysteine levels: 1. Vitamin B6 (PLP) 2. Choline (may raise methionine levels, which reforms homocysteine) 3. Folate (via methionine synthase) (Vitamin B12 is usually not lacking) 4. Different organs use amino acids differently a. Most AA are deaminated and the nitrogen is used for urea synthesis b. Liver receives AA from the diet (via the gut) and delivers to muscle
i. Is the first organ that metabolizes a significant amount of the amino acids from digestion (from the intestine), but is low in the branched-chain amino transferases

c. Muscle takes up the AA from the blood not used by liver i. Branched chain amino acids (BCAA) isoleucine, leucine, and valine are preferentially metabolized as fuel in muscle (where their transaminases are primarily localized) 1. Carbon skeletons are oxidized to give ATP 2. NH3 ends up as Glutamine or Alanine and leaves the muscle a. Alanine is transaminated to pyruvate in the liver and serves as a substrate for gluconeogenesis i. The resulting glucose can be used in the Cahill cycle (to regenerate alanine), which provides a 17

mechanism for transporting nitrogen from degradation of muscle protein back to the liver, and the recycling of pyruvate from the liver for fuel for muscle

b. Glutamine i. Liver maintains levels of Glutamine in the blood ii. Plasma glutamine is an important carrier of nitrogen, carbon, and energy between organs, and can be used for urea synthesis, ammoniagenesis, gluconeogenesis, and respiratory fuel iii. Used by the intestine for fuel: A solution of glutamine is used to promote GI tract healing and nutritional supplementation with GI disorders, HIV/AIDS, cancer, and other critical illnesses iv. The kidneys use glutamine for ammonia production using the glutaminase reaction, especially when pH control is needed

32. Purine (A/G) Metabolism

1. Nucleotides have three components: a. Nitrogenous base i. Purine: adenine & guanine ii. Pyrimidine: thymine & cytosine b. Five-carbon sugar i. Ribose ii. 2deoxyribose iii. Nucleoside = base + sugar c. Phosphate groups Nucleotides have critical roles in important biochemical events: a. Nucleotides are activated precursors of DNA & RNA b. Nucleotides are components of 3 major coenzymes (NAD+, FAD and CoA) c. ATP is a universal currency of energy d. GTP powers movement of macromolecules (e.g. activation of Ras signaling) e. Nucleotide derivatives are activated intermediates in many biosynthesis events i. UDP-glucose glycogen ii. CDP-diacylglycerol phosphoglycerides

2.

3. Purine nucleotide biosynthesis a. De novo synthesis (using small precursors in cytosol of liver or placenta) i. Pentose-phosphate shunt: can use glucose-6-phosphate for synthesis of ribose-5phosphate 1. R5P + ATP 5phosphoribosyl-118

pyrophosphate (PRPP) [via PRPP synthetase] 2. Next is replacement of the pyrophosphate on PRPP with the amide group of glutamine to form 5-phosphoribosylamine a. Committed step in purine nucleotide biosynthesis; is when the glycosidic bond is formed as the first atom of the purine ring is incorporated b. Enzyme: glutamine phosphoribosyl pyrophosphate amidotransferase (PRPP Formation of Inosinate amidotransferase) i. Is inhibited by the end products of purine biosynthesis ii. Formation of inosine monophosphate 1. The first purine synthesizes is inosinic acid (IMP)
2. Purine ring is assembled around the amino group of phosphoribosylamine until the ribonucleotides inosinic acid (IMP) is produced Four ATP molecules are used

3.

iii. Conversion of IMP to AMP and GMP 1. Inosinic acid serves as a precursor for both AMP and GMP 2. Synthesis of AMP (from IMP) requires GTP; synthesis of GMP requires ATP thus, an abundance of one purine nucleoside triphosphate ensures the production of the second; this arrangement helps to insure equal levels of purines iv. Conversion of monophosphates to diphosphates and triphosphates 1. Conversion of Monophosphates to Diphosphates a. This conversion is catalyzed by kinases that are basespecific but not sugar-specific (e.g. GMP kinase) 2. Conversion of Diphosphates to Triphosphates a. Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase b. Enzyme has broad specificity v. Activation of Antiviral Nucleoside Analogues 1. Many nucleotide analogues require the formation of their corresponding 5 triphosphates (TP) 2. For example, in order to function AZT must be converted to AZT triphosphate 3. AZT triphosphate is formed by the sequential action of thymine kinase (nucleoside), thymidylate kinase and nucleotide diphosphate kinase b. The salvage pathway (extrahepatic tissues)
i. Free purines can be used to make new nucleotides via salvage pathways which permit efficient reutilization of preformed purine bases (derived from sources such as the breakdown of nucleic acids)

ii. Two salvage methods: 19

iii. Direct conversion of a base into a ribonucleotides 1. Hypoxanthine- guanine phosphoribosyl transferase (HGPRT) a. Guanine (Base) + PRPP GMP + PPi b. Hypoxanthine (Base) + PRPP IMP + PPi 2. Adenine phosphoribosyltransferase (APRT) a. Adenine (Base) + PRPP AMP + PPi iv. Reversible conversion of bases to nucleosides and then nucleotides 1. Purine nucleoside phosphorylase a. Nucleoside N + Pi base + ribose-1-phosphate
2. *Rarely used to salvage a base; the predominant direction of the reaction catalyzed by this enzyme is derivative Nucleoside kinase a. Nucleoside N + ATP NMP + ADP b.

4. Catabolism of purine nucleotides a. Conversion of nucleotides to nucleosides i. AMP Adenosine (nucleoside) + Pi (via hydrolysis by phosphatases) b. Adenosine deaminase reaction i. Substrates: adenosine, ribose, deoxyribose c. Purine nucleoside phosphorylase (a nucleosidase)
i. Yields free base and ribose-1-phosphate ii. Reaction is reversible, but the concentration of free purine and R1P are generally too low to support synthesis via the salvage pathway

d. Uric Acid Formation i. Hypoxyanthine: primary breakdown product of AMP via adenosine and inosine; is oxidized to xanthine, then to Control Pathways for Purine Biosynthesis uric acid (via xanthine oxidase in both steps) ii. Guanine (breakdown product of GMP) is converted to xanthine by guanine deaminase 5. Regulation of purine biosynthesis a. De novo purine biosynthesis regulation occurs at: i. PRPP synthetase reaction 1. Activity depends on intracellular concentrations of several end products (AMP, GMP, ADP, etc) of which PRPP is a substrate ii. Amidophosphoribosyltransferase reaction
1. Salvage pathways generate AMP and GMP through APRT and GHPRT phosphoribosyltransferase reactions

Salvage shuts off de novo pathway

2. Salvage pathways shut off the de novo pathways at the PRPP amidotransferase step 3. AMP & GMP are feedback inhibitors
4. The amidotransferase has separate allosteric sites for these two nucleotides PRPP is consumed during the formation of purines in the salvage pathway, which decreases the rate of formation of 5-phosphoribosylamine a.

amidotransferase
phosphoribosyltransferase (salvage)

PRPP Conc.

20

iii. Branch point in the formation of AMP and GMP from IMP 1. AMP & GMP negatively regulate their respective formation from IMP 6. Disorders of purine nucleotide metabolism a. Gout: elevated uric acid levels in the blood, due to a variety of metabolic abnormalities
i. Although high levels of uric acid may defend against aging and cancer caused by oxidants and free radicals, too high a level leads to gout

ii. Urate crystals are deposited in the cartilage of joints resulting in pain and disability (also deposited in the kidney producing renal damage)
iii. Can be due to: 1. Overproduction of purines through defective control mechanisms 2. Failure to excrete uric acid (due to renal function impairment)

iv. Enzyme defects that lead to Gout: 1. A partially defective HGPRT a. Causes reduced IMP & GMP formation via the salvage pathways; PRPP accumulates and causes activation of de novo purine biosynthesis b. The lower IMP & GMP levels result in reduced feedback inhibition of the de novo pathway 2. PRPP synthetase enzyme (increased activity)
a. Is less susceptible to feedback inhibition by purine nucleotides

b. The increased enzyme activity leads to overproduction of PRPP, and thus activation of the de novo pathway 3. Glucose-6-phosphatase enzyme a. Enzyme deficiency leads to increased utilization of the pentose-phosphate pathway, and consequently, to excessive production of ribose-5-phosphate, the immediate precursor of PRPP v. Allopurinol: lowers uric acid levels in the blood 1. Analogue of hypoxanthine 2. Competitive inhibitor of xanthine oxidase (converts hypoxanthine
& xanthine to uric acid)

3. Leads to accumulation of hypoxanthine & xanthine, which are more soluble than uric acid and more easily excreted b. Lesch-Nyhan syndrome i. Infants with this disease lack a functional HGPRT ii. Marked increase in the rate of purine biosynthesis by the de novo pathway iii. Symptoms include self-mutilation, mental illness and gout like symptoms owing to elevated uric acid in serum
iv. The mechanism by which the deficiency of HGPRT causes central nervous system disorders remains unknown

1. However, in normal subjects, HGPRT activity is highest in the brain, suggesting the importance of the purine salvage pathway in this tissue
v. Allopurinol is used to treat the Gout but there is currently no cure for the neurological problems

21

c. Adenosine deaminase deficiency i. Causes one form of severe combined immunodeficiency (SCID): patients lack both B and T lymphocyte functions and are usually dead from infections before age 2
ii. Mechanism is unknown but related to buildup of dATP (inhibitor of ribonucleotides reductase, and ultimately of DNA synthesis); lymphocytes are particularly sensitive to excessive levels of dATP

7. Inhibitors of purine biosynthesis a. Sulfonamides

i. First antibacterial agents employed in humans; bacteria must synthesize folic acid compounds; mammals depend on preformed folate

ii. Sulfonamides function as an analog of p-aminobenzoic acid (a component of folic acid) and inhibit synthesis of folate in bacteria
1. Folate is required in de novo biosynthesis (and also for thymidylate and methionine biosynthesis)

iii. Blocks the formation of an essential bacterial compound and thus, their growth b. 6-Mercaptopurine i. Functions as an analogue of hypoxanthine 1. Is converted to a nucleotide by HGPRT, which draws some of the PRPP away from nucleotide biosynthesis pathways
2. The 6-mercaptopurine nucleotide accumulates in the cell and functions as an analog of purine nucleotides

ii. Prevents the production of AMP and GMP (after salvage, is competitive inhibitor of IMP pathways for AMP & GMP biosynthesis) iii. Used in the treatment of acute leukemia (antitumor drug)

33. Pyrimidine (T/C) Metabolism

1. Synthesis of pyrimidines a. Pyrimidine skeleton is assembled first and is subsequently attached to PRPP b. A major regulatory step is formation of carbomyl phosphate c. De Novo Synthesis: d. Step I: Carbamoyl phosphate synthase II i. Takes place in the cytoplasm and IS NOT part of the urea cycle (urea cycle takes place in liver mitochondria and is catalyzed by a different ii. Glutamine + 2 ATP + HCO3- Carbamoyl phosphate + 2 ADP + Glutamate e. Step II: Aspartate Transcarbamoylase i. Carbamoyl phosphate condenses with aspartate f. Step III: Synthesis Of Orotate i. The ring is closed in the dihydroorotase reaction
enzyme)

22

ii. Dihydroorotase dehydrogenase makes orotate, the immediate precursor of all pyrimidines iii. All three steps take place on one protein with multiple enzymatic activities g. Step IV: Synthesis of UMP i. Orotate condenses with PRPP to form orodylate ii. UMP is next generated by a decarboxylation reaction h. Step V: UTP Formation i. Uridylate kinase: UMP + ATP UDP + ADP ii. Nucleoside diphosphate kinase: UDP + ATP UTP + ADP i. CTP is formed by amination of UTP

j. Salvage pathway: k. Pyrimidine salvage is possible via the reversible conversion of bases to nucleosides catalyzed by a phosphorylase 2. Pyrimidine catabolism a. In contrast to purine catabolism, pyrimidine catabolism (which occurs mainly in the liver) yields soluble end products b. Nucleotides are first converted to nucleosides by phosphatases c. Cytidine is converted to uridine by cytosine deaminase d. Uridine and thymidine (nucleosides) are converted to free bases by pyrimidine nucleoside phosphorylase i. Catabolism of uracil and thymine (bases) proceed in parallel steps; catalyzed by the same enzymes ii. Large numbers of cells are killed, and DNA degraded, in cancer patients undergoing chemotherapy iii. It is possible to estimate the turnover of DNA by measuring baminoisobutyrate (end product of thymine pyrimidine catabolism) 3. Synthesis of Deoxyribonucleotides: Ribonucleotide Reductase a. All four ribnucleoside diphosphates (ADP, GDP, CDP, UDP) can be converted to the deoxyribonucleoside by ribonucleotide reductase b. The electron donor for this reaction is NADPH via oxidation of the protein thioredoxin i. The sulfhydryl groups are regenerated by a reaction with NADPH catalyzed by thioredoxin reductase ii. Hydroxyurea (an antineoplastic agent and inhibitor of DNA synthesis) inactivates the enzyme ribonucleotides reductase 23

4. Control mechanisms for nucleotide formation a. Mechanism of Ribonucleotide Reductase i. Two sulfhydryl groups on ribonucleotide reductase reduce the ribonucleoside to the deoxyribonucleoside ii. Thioredoxin sulfhydryls regenerate the oxidized sulfhydryls on ribonucleotide reductase iii. Thioredoxin reductase regenerates the sulfhydryls on thioredoxin using NADPH b. Regulation of de novo pyrimidine metabolism i. In mammalian cells, regulation occurs primarily at the level of carbamoyl phosphate synthetase II ii. Next level of regulation is at the level of OMPdecarboxylase 1. UMP is an inhibitor of OMP-decarboxylase c. Control of biosynthesis of dNTPs i. The reduction of ribonucleotide diphosphates is controlled by allosteric interactions 1. E.g binding of dATP to allosteric sites on ribonucleotide reductase renders the enzyme inactive 2. Alternatively, binding of ATP activates the enzyme ii. Regulation permits a fine adjustment of the dNTP pools required for DNA synthesis 5. Pyrimidine analogues a. Synthesis of Thymidine i. TMP is formed by the methylation of dUMP ii. Reaction catalyzed by thymidylate synthase iii. N5, N10-methylene tetrahydrofolate is the one-carbon donor

24

b. Nucleotide metabolism as targets for chemotherapy i. Thymidylate synthase inhibitor: Fluorouracil ends up being covalently attached to the enzyme, essentially killing the enzyme (suicide inhibitor) ii. Dihydrofolate reductase inhibitor: Aminopterin and Methotrexate 1. Methotrexate is a Folate Analogue and a Competitive Inhibitor of Dihydrofolate Reductase 6. Coordination of purine and pyrimidine nucleotide biosynthesis a. PRPP activates carbamoyl phosphate synthetase II and is a rate limiting substrate for orotate phosphoribosyltransferase. b. Recall that PRPP activates amidophosphoribosal-transferase and that PRPP is the substrate for HGPRT and APRT

34. Integration of Metabolism

What happens when you eat?

1. Food and fuel (or fat) a. Storage i. 400 gm (1700 cal) Glycogen ii. 6,200 gm (24,000 cal) Protein iii. 15,000gm (138,800 cal) and up Triglycerides b. Energy: Variable demand i. 1,400 (couch potato) ii. 3450 calories (runner) iii. 9,000 (severe burns; infection) C/day 2. Fuel transport between organs

Food

Circulation

a. Brain uses majority of glucose - 120g /day, which is 60% or more of the total 25

b. Liver produces ketone bodies or FA for muscle heart and brain c. Lactate from muscle or RBS comes back to liver to make glucose d. Liver maintains the glucose levels; can use fatty acids and glycerol to make glucose e. Type I diabetes & starvation: liver can generate ketone bodies (source of energy due to excess acetyl CoA) 3. Different organs work differently Tissue Liver Storage Fuel Glycogen Triacylglycerol (TG) Glycogen Preferred Fuel Glucose Fatty acids (FA) Amino Acids FA Glucose TG FA FA Glucose (Ketones in starvation)
Protein Carbohydrate GLU-6-P
Glucose-6-P NADPH TG PL TG Glycerol-3-P

Exported Fuel Glucose FA Ketones

Skeletal Muscle (resting) Skeletal Muscle (working) Adipose Heart Brain

Alanine Lactate FA (heart) Glycerol (liver)

4. Links between metabolism of carbohydrates, fats and proteins

Carbohydrate Fat Fat Carbohydrate

Carbohydrate

Protein
Trp Ala Gly, Ser Cys Pyruvate

3-P-glycerate
Glucose

Ser, Gly, Cys His Arg Glu Gln a-ketoglutarate

DHAP

Glycerol-3-P

Pyruvate

Acetyl CoA TG Fatty acids PL Ketone bodies Cholesterol

Pyruvate a-ketoglutarate Oxaloacetate

Ala Glu, Gln, Pro

Pro Thr Met Succinyl CoA Val Phe Asp Tyr Fumarate Oxaloacetate Ileu

Asp, Asn

5. Regulating individual steps in metabolism 26

a. Allosteric inhibition or stimulation i. Rapid response; results in marked changed in concentration in enzyme activity may result from only a moderate change in its regulators concentration
EXAMPLES OF ALLOSTERIC REGULATION
All from the well-fed state Glucose: Phosphofructokinase I - By F2,6 BP to Drive Glycolysis Fructose 1,6 BPase By F2,6 BP to Inhibit Gluconeogenesis Fatty Acids: AcetylCoA Carboxylase- By Citrate to Make Fat. Pyruvate kinase - by both F-1,6 P2 and F2.6 P2 to Make Fat. Carnitine Acyl Transferase 1 By Malonyl CoA to Block FA breakdown. Glycogen metabolism: Glycogen Synthase - By Glucose 6 Phosphate to Make Glycogen Glycogen Phosphorylase By Glucose to Decrease Glycogen Breakdown

The most important point remains that you can guess what the effect should be based on common sense Citrate in excess says that you have plentiful CHO: would like to store energy Malonyl CoA says you are making fat: dont want to break it down so you block transport G6P says that you have plentiful CHO and would like to store it Same token, plenty of glucose means you dont want to make more by breaking down glycogen

Enzyme Pyruvate Kinase Acetyl CoA carboxylase Pyruvate Dehydrogenase Glycogen synthetase

Allosteric activation in the well-fed state + Effector Effect F1,6BP & F2,6BP Citrate Pyruvate Glucose-6-P Provides pyruvate for fat synthesis Stimulation of fat synthesis Provides acetyl CoA for fat synthesis Increases storage of carbohydrate as glycogen Provides pyruvate for fat synthesis

Activated Pathway Glycolysis FA Synthesis FA synthesis Glycogen synthesis

Phosphofructokinase

F2,6BP

FA synthesis

Allosteric inhibition in the well-fed state Enzyme Glycogen phosphorylase Carnitine acyl transferase-1 Phosphofructokinase Fructose 1,6 BPhosphatase - Effector Glucose Malonyl CoA Cytoplasmic citrate F2,6BP Gluconeogenesis 27 Effect Decreases glycogen breakdown Decreases FA breakdown Inhibited Pathway Glycogen breakdown FA breakdown

b. Regulation of the amount of enzyme i. Inhibition or stimulation of gene transcription ii. Protein degradation or stabilization iii. High protein: 1. Induction of enzymes needed for nitrogen metabolism a. Ornithine carbamoyl transferase b. Argininosuccinate synthetase c. Argininosuccinase 2. AA catabolism/ gluconeogenic a. Serine dehydratase b. Tyrosine aminotransferase c. Alanine aminotransferase d. Cystathionase e. Glutaminase f. Ornithine aminotransferase iv. High carbohydrate: 1. Glucose uptake a. Amylase (for breakdown) b. Glucokinase (to fix glucose for glycogen synthesis) 2. PPP a. Glucose-6-phosphate dehydrogenase b. 6 phosphogluconate dehydrogenase 3. Fat biosynthesis (to store excess) a. Acetyl CoA carboxylase b. Fatty acid synthetase c. NADP-malate dehydrogenase d. Citrate lyase (acCoA) v. High Fat: 1. FA breakdown a. Lipase (to break down lipids) b. Carnitine palmitoyl transferase (to transport FA) 2. Inhibit PPP (NADPH) a. Glucose-6-phosphatase 3. Glucose production a. Glucose-6-phosphatase b. Serine dehydratase (pyr) c. Ornithine aminotransferase (proline) d. Tyrosine aminotransferase e. Fructose bisphosphatase

28

Induced enzymes in well-fed state Glucose uptake for storage Glucokinase PPP provides NADPH for fat synthesis G6P dehydrogenase 6-phosphogluconate dehydrogenase Glycolysis provides pyruvate for fat Pyruvate kinase synthesis Provided NADPH for fat synthesis NADPH- linked malate dehydrogenase Provides cytoplasmic acetyl CoA for fat Citrate lyase synthesis Increases FA synthesis Acetyl CoA carboxylase Fatty acid synthase Increases cholesterol synthesis HMG CoA reductase

Induced enzymes in starvation state Increases glucose relative to blood Glucose-6-phosphatase Pyruvate carboxylase Increases gluconeogenesis PEP carboxykinase Various amino acid transaminases c. Covalent modification i. Inhibition or activation by phosphorylation 1. Phosphorylation can change Km, Vm or localization 2. Balances between gluconeogenesis and glycolysis ii. Dephosphorylation: an insulin effect in the well-fed state (a state where you need to absorb glucose) 1. Enzymes activated by dephosphorylation are for: a. Glycogen storage 29

b. FA synthesis c. Cholesterol/ TAG synthesis 2. Enzymes inhibited by dephosphorylation are for: a. Glycogen breakdown b. Gluconeogenesis iii. Phosphorylation of Hormone-Sensitive Lipase in presence of glucagon 1. Allows triglycerides to be broken down to free fatty acids and glycerol 2. Insulin leads to HSLs dephosphorylation and inhibition of the breakdown of triglycerols & fatty acids d. Compartmental separation
Muscle Regulation of Fatty Acid Oxidation Via Compartmentalization

MITO
Fatty Acid Oxid

CPT -I

Fatty-Acyl CoA Acetyl CoA Malonyl CoA MDC ACC-2 Acetyl CoA

AMPGK AMP-PK

6. Hormonal control of metabolism

LOW BLOOD GLUCOSE

Hypothalamic Reg Ctr Pituitary ACTH Autonomic Nervous System Pancreas Adrenal Cortisol Glycogenolysis Gluconeogenesis 0 ++ Epinephrine Norepinephrine Glucagon +++ 0 +++ 0 ++ ++

a. Mechanisms of hormone action i. G-Protein/ Cyclic AMP systems 1. Hormones: glucagon, epinephrine 2. All Receptors That Activate cAMP Have Similar Overall Structure a. They all span the membrane 7 times b. Highly hydrophobic transmembrane region

Adenylate cyclase (cAMP) Cascade Receptor G protein Adenylate cyclase cAMP

Protein Kinase A Phosphorylation of enzymes, transcription factors, et cet.

30

c. All have the same intracellular domains (same Gprotein) but different extracellular domains to provide specificity 3. Mechanism: 4. Hetero-trimer inhibitory subunits and GTP/GDP binding subunits 5. Receptor (Guanine nucleotide exchange factor, GEF) causes dissociation of subunits and GDP release allowing GTP to take its place 6. GS- then activates adenylate cyclase 7. GS- then hydrolyzes bound GTP to GDP with the help of 1. GTPase activating proteins (GAPs) shutting of the process 2. GDP Gs then binds again 3. Adenylate Cyclase Synthesizes cAMP (and Phosphodiesterase degradation of cAMP) a. Caffeine inhibits the cAMP phosphodiesterase enzyme 4. cAMP activates Protein Kinase A a. Promotes dissociation of regulatory (R) subunits and liberation of Catalytic (Cat) subunit b. PKA regulates both glycogen breakdown and synthesis
PKA Regulates Both Glycogen Breakdown and Synthesis
Glucagon

R
G-GTP Adenylate cyclase

Protein kinase A Cat-Reg

Protein kinase A Cat

+ Reg-cAmp

Phosphorylase kinase

P-Phosphorylase kinase Glycogen synthase P-Glycogen synthase

Phosphorylase

P-Phosphorylase

More glycogen breakdown

Less glycogen synthesis

ii. Tyrosine Kinase Insulin receptor sets off a signaling cascade iii. Steroid hormones (Cortisol) bind in the cytoplasm and then move into the nucleus 1. Binds intracellular receptors and promotes glucose synthesis 2. Direct transcriptional activators 7. Starvation a. 1st Phase: well-fed phase that lasts 4 hours while exogenous fuel is supplying and maintaining blood glucose levels i. Gluconeogenesis starts 4-6 hours after food and becomes fully activated as glycogen is depleted ii. Carbon skeletons are derived from lactate, glycerol and amino acids b. 2nd: Post-absorptive state (6-16 hours after a meal/ overnight fast) i. Liver glycogen breakdown to get glucose begin switch to FA for fuel 1. Liver glycogen is used up by 18 hours into fast 31

Stages of Starvation

c. 3rd: Early starvation (16- 48 hours) i. Gluconeogenesis from AA: thus protein breakdown begins increase in urea excretion ii. Glucoconeogenesis is elevated iii. Triacylglycerols produce FAs for energy; glycerol for Origin of Hepatic Hepatic and Glycogen additional glucose production Blood gluconeorenal glucoExogenous Hepatic Glucose genesis neogenesis gluconeoiv. Muscle decreases use of Glycogen genesis Tissues All except liver. Brain, RBC, glucose, increases use of FA Using All All except liver. Muscle and fat renal medulla Glucose Muscle and fat tissue at rate Small amt by 1. Muscle glycogen not tissue at between III and muscle decreased rate IV useful for anything but muscle d. 4th: Intermediate starvation (2- 24 days) i. Muscle mass decreases as AA go to glucose ii. FA are broken down to ketone bodies for use by brain 1. Decrease in gluconeogenesis iii. Renal and hepatic gluconeogenesis to increase blood glucose e. 5th: Prolonged starvation (24- 40 days) i. Brain switches to ketone bodies ii. Glucose is still produce by liver and kidney gluconeogenesis, and is used mainly by RBCs and the brain iii. Total amount of nitrogen excreted as urea decreases dramatically to save protein. iv. Urinary ammonia initially increases to conserve cations to prevent ketone body excretion v. Blood metabolite levels: 1. Increase in: a. Ketone bodies b. Free fatty acids 2. Decrease in: a. Glucose b. Urinary nitrogen 3. Urinary ammonia initially increases and then decreases 4. Urea increases due to protein breakdown to synthesize glucose, and then decreases later on to preserve glucose (protein)
FUEL METABOLISM DURING EARLY STARVATION (16-32 HRS) TISSUES BLOOD
KETONE BODIES ATP + CO2

Hepatic and renal gluconeogenesis Brain at decreased rate, RBC, renal medulla

FUEL METABOLISM DURING MIDDLE AND LATE STARVATION TISSUES

ATP + CO2

LIVER

URINE

BLOOD
KETONE BODIES

LIVER

FATTY ACIDS GLYCEROL AMINO ACIDS

KETONE BODIES

FATTY ACIDS STORED FUELS GLYCEROL AMINO ACIDS

KETONE BODIES

STORED FUELS Triglycerides (160 g/day) Protein (75 g/day) GLUCOSE LACTATE ATP + CO2

Ac-CoA

ATP + CO2

Triglycerides (160 gday) Protein (20 g/day) GLUCOSE LACTATE ATP + CO2

Ac-CoA

ATP + CO2

GLUCOSE LACTATE

GLUCOSE LACTATE

32
GLUCOSE

GLUCOSE

f. Three factors keep blood glucose constant: i. Mobilization of glycogen; gluconeogenesis ii. Release of free fatty acids 1. As the fast sets in, fatty acids are mobilized and liver switches over to using them as fuel 2. Fat is mobilized by action of hormone sensitive lipase and aided by catecholamines, epinephrine and especially norepinephrine a. In fasting lipoprotein lipase activity is low, so circulating TAG not available to make fat 3. Fatty acids are released into the blood; transported bound to albumin iii. Switch of muscle and liver to using free FA as fuel
Glucose metabolism in liver
A. After a meal

Fuel Flow During an Overnight Fast

B. After overnight fast

EXTRA SLIDES:
Interplay of Urea Cycle and Gluconeogenesis

Interplay of Urea cycle and gluconeogenesis The relationship of aa catabolism to gluconeogenesis. Urea Cycle: Get OAA, fumarate for gluconeogenesis

33

Fate of Glucose in the Cell

PPP

Fate of Acetyl CoA

Fatty Acid Biosynthesis TCA

Ketone Bodies In case of excess acetyl CoA in starvation, (i.e no OAA for TCA cycle due to glucose depletion. Then ketone bodies are produced

35. Diabetes
1. Diabetes as a Disease 34

a.

Facts & Figures: i. 23.6 million people (7.8%) have diabetes. ii. 23.1% of people older than 60 have diabetes. iii. Estimated lifetime risk: as high as 30-40% (somewhat higher for women) iv. Overall risk of death: Twice that of non-diabetics at any age v. Total Cost (direct and indirect): $174 billion/yr vi. Medical Expenses 2.3X higher than non-diabetics

Characteristics of the Two Major Types of Diabetes

b. Diabetes = Starvation in the presence of plenty c. Type 1 (5-10%): loss of cells (other: Gestational, i. Little or no Maturity Onset Diabetes of the Young (MODY) circulating insulin beta-cell dysfunction) ii. Normal response to administration of insulin iii. Often present with ketoacidosis d. Type 2 (~90%): insulin resistance/ cell failure i. Older onset ii. Cells don't respond to insulin iii. Generally don't show ketoacidosis 2. Insulin Synthesis and Secretion a. Insulin is produced by processing of precursors (preproinsulin and proinsulin) b. Insulin production and secretion is controlled by: i. Glucose 1. High Glucose G6P ATP closes K channel Ca secretion insulin secretion 2. Insulin Secretagogues block potassium channels causing depolarization a. Close the ATP-sensitive K+ channel leading to a rise in intracellular calcium, increased fusion of insulin granulae with the cell membrane, and therefore increased secretion of (pro)insulin b. Sulfonylureas; Meglitinide 3. Metformin reduces hepatic glucose output a. Inhibits gluconeogenesis (decreases BGL) ii. Incretins: hormones produced in the digestive tract 1. Increases insulin release when orally-ingested glucose levels are normal or elevated a. Dont act when glucose levels are low 2. GIP = glucose-dependent insulinotropic peptide 3. GLP = glucagon-like peptide a. Acts on secretion via ion channels 35

b. Blocking this breakdown enhances insulin secretion 4. Exenatide: GLP1/GIP analogue 5. Sitagliptin (Januvia): DPPIV protease inhibitor that stabilizes GLP1/GIP protein a. Blocks the degradation of incretins 3. Insulin Signaling a. Tyrosine kinase pathway i. Insulin establishes a cascade with a lipid second messenger b. Insulin induces glucose uptake by inducing glucose transporters in fat and muscle i. Induces Glut4 activity and localization
How Does Insulin Signal?? Insulin Establishes Multiple Signal Cascades
Lipid Kinase
Insulin Receptor is a tyrosine kinase Cell Membrane Lipid 2nd Messenger:PIP3Phosphatidylinositol 3,4,5trisphosphate
Ras

2nd Messenger:PIP3Phosphatidylinositol

Ras

Adaptor

Adaptor Protein kinases Protein kinases

Protein kinases Alterations in transcription

A Protein Phosphatase is one important target.

Transcription

Alterations in transcription

c.

In the liver: the major transporter is glut 2; gluckokinase traps the glucose by phosphorylation. This has a higher Km than hexokinase and is an adaptive response to high carbohydrate

4. Insulin and Metabolism a. After eating: i. Glucose level goes up ii. Insulin level also goes up because glucose stimulates beta cells to secrete insulin 1. Much less increase in insulin after a protein rich meal iii. Glucagon goes down after a meal balanced with CHO (glucagon is produced by alpha cells); insulin flows past alpha cells where it inhibits glucagon secretion b. Insulin/glucagon ratio (I/G) ratio is a determining factor in balancing metabolism c. Diabetics have poor glucose control that can be seen in fasting glucose levels of glucose tolerance tests i. Insulin/glucagon ratio is low in diabetes ii. Low Insulinglucose is not absorbed 1. Delay in glucose absorption or decrease in plasma glucose iii. Glucagon stimulates glycogenolysis (initially) AND gluconeogenesis

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iv. Result: Higher baseline for resting blood glucose d. Insulin acts to reduce blood glucose Insulin Acts to Reduce Blood Glucose in 3 Major Ways i. Promotes: ADIPOCYTES 1. Glycogen synthesis VLDL LIVER 2. Glucose uptake into TRIACYLGLYCEROLS GLYCOGEN tissues FATTY ACIDS GLYCOGEN + FATTY ACIDS 3. FA biosynthesis and GLUCOSE KETONE BODIES storage as triacylglycerol KETOSIS Increases 4. Glycolysis in the liver & glucose transporters GLUCONEO (e.g fat and muscle). UPTAKE acetyl CoA formation LACTATE GLUCOSE AMINOACIDS + 5. LPL and lipid transfer to GLYCOGEN adipocytes KIDNEY, dehydration 6. Amino acid uptake into SKELETAL MUSCLE cells increases protein synthesis ii. Inhibits: 1. Glycogenolysis (glycogen phosphorylase) 2. Gluconeogenesis 3. HSL (therefore, increasing lipid deposition) 5. Insulin Affects Carbohydrate and Fat Metabolism a. decreases blood glucose by increasing uptake in muscle and adipose cells b. decreases glycogen phosphorylase and increases glycogen synthetase activity; c. decreases gluconeogenic reactions; d. increases glycolysis in the liver, increasing acetyl CoA formation; e. increases fatty acid synthesis in the liver f. increases lipoprotein lipase, increasing lipid transfer to adipocytes; g. increases glucose in adipocytes, increasing triacylglycerol synthesis; h. decreases hormone-sensitive lipase, increasing lipid deposition i. increases amino acid uptake into cells, increasing protein synthesis 6. Insulin resistance a. Results from: i. Genetics (e.g. IRS-1 mutation) ii. Changes in free fatty acid levels iii. Proinflammatory cytokines by macrophages iv. Alterations in adipokines (e.g adiponectin and resistin) 1. Resistin: confers insulin resistance 2. Adiponectin increases sensitivity v. Regulating Sensitivity to Insulin Signaling b. Loss of Insulin: i. Increase glycogen breakdown to increase glucose 1. Phosphorylate and stimulate glycogen phosphorylase for breakdown 2. Phosphorylate and inhibit glycogen synthase for synthesis ii. Net increase in gluconeogenesis iii. Increase lipolysis (no phosphorylation of hormone sensitive lipase)
+

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1. FAs give increased triglycerides, but ketone bodies are the cause of diabetic ketosis 2. Increases TG, increases a form of LDL, decreases HDL a. Hypertriacylglycerolemia i. The lipid triad: elevated triglycerides, low HDL, small, dense low-density lipoprotein (LDL) c. Hyperglycemia: i. Results from loss of insulin ii. Without insulin, GLUT4 is not translocated to the plasma membrane of fat and muscle cells and glucose uptake is impaired iii. Glucose reacts with metals to generate reactive oxygen species (ROS) iv. The polyol pathway generates sorbitol and affects the NADPH/NADP+ ratio 1. NADPH is primarily used for biosynthesis; hyperglycemia affects biosynthesis pathways rates v. Advanced glycation end products (AGE) contributes to vascular damage
1. 2. Elevated glucose gives protein glycation (stable attachment) Hb glycation gives carbohydrate history (Average level of CHO over lifespan of Hb) a. Hemoglobin A1c integrated history: tells you well how well the patient is controlling the disease b. Normal HbA1c = 4-6% c. Diabetic 7-8%

7. Complications of diabetes a. Diabetic RETINOPATHY b. Diabetic NEUROPATHY

Diabetic ketoacidosis (DKA): this coma is a medical emergency 1. Intravenous fluids, insulin, and administration of potassium and sodium ii. Hyperosmolar coma: plenty of intravenous fluids, insulin, potassium and sodium given as soon as possible iii. Hypoglycaemic coma: administration of the hormone glucagon to reverse the effects of insulin, or glucose given intravenously i.

c. Diabetic NEPHROPATHY i. 30-50% develop kidney disease, proteinuria ii. Kidneys respond to high levels of blood glucose by doing their best to excrete it, along with a great deal of water d. Cardiovascular complications i. Coronary artery disease risk 4 times greater, hypertension
FUEL METABOLISM DURING SEVERE UNTREATED DIABETES TISSUES
ATP + CO2 FATTY ACIDS STORED FUELS Adipose Fat Muscle Protein AMINO ACIDS GLUCOSE GLUCOSE LACTATE ATP + CO2 GLUCOSE LACTATE GLYCEROL KETONE BODIES

URINE

BLOOD
KETONE BODIES

LIVER

Ac-CoA

ATP + CO2

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