Exam IV Review

8/12/2019 Exam IV Review

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BIOCHEMISTRY

28. Protein Digestion

1. Amino acids in excess of requirements aredegraded to products that are either oxidizedfor energy or converted into carbohydratesor fats

2. Amino acids are maintained at certain levelsin the blood for use by the body

a. Amino acid metabolism =NITROGEN metabolism

b. These amino acids come from twosources: our own body breakdown(about ) and the digestion ofdietary protein (about )

c. Major amino acids in blood:Alanine and Glutamine

3. Dietary protein cannot be absorbed intactand is first hydrolyzed to its constituent amino acids (stomachsmall intestineliver (via portal vein))

4. Three stages of protein digestion:a. Gastric (pH 1.5)

i. Glands of the stomach lining secrete acidand pepsinogenii. Acid denatures proteins; pepsinproduces peptides

1. Pepsinogen is not active at pH > 2due to a pro-peptide(prosegment) at the active site; autoactivation at pH < 2results in active pepsin

iii. Peptides enter the small intestineb. Intestinal (pH 6-8)

i. Acid chime enters duodenum where proteolysis occursii. Partially digested protein triggers the release of hormones (secretin

and cholecystokin) from specialized mucosal endocrine cellsiii. Results in contraction of gallbladder and release of an alkaline

secretion from pancrease containing bicarbonate and a mixture ofendopeptidases (trypsin, chymotrypsin, and elastase; a.k.a serineproteases) and the exopeptidases (carboxypeptidase A and B)

1. Endopeptidases: hydrolyze internal peptide bonds2. Exopeptidases: snip off N or C terminal residuesiv. Enzymes in the duodenum digest long peptides into short ones

v. Enzymes of intestinal lumen (aminopeptidases and dipeptidases)produce mostamino acid

vi. Enzymes inside intestinal cells hydrolyze to amino acids bloodc. Pancreatic

i. Pancreas secretions initially produce inactive proteases (bicarbonate &zymogens)

Body Protein

220 g/d

Amino Acid Pool

Synthesis

Nitroge

containi

Compoun

Dietary Protein

70-100g/d

Feces

10g/d

Protein Synthesis

In Balance (digestive enzymes+body protein):

about 300 g/d

Degradation for energ

or to make glucose

Digestive Enzymes

70-100g/d

Sources and Uses of Amino Acids

The body maintains a pool of amino acids in the blood stream and tissues forprotein synthesis, to use as fuel, or for conversion into other substances.

The ONLY significant source of nitrogen in our diet is through protein


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1. Trypsinogen(from pancreas)(via enteropeptidase)trypsin2. Enzyme enteropeptidase(secreted from small intestine) is the

key event in activation of trypsinogen; conformation changea. Secreted from the luminal surface of the small intestine

under the influence of CCK

b.Newly formed trypsin acts autocatalyticallybyhydrolyzing the same bond as enteropeptidase

c. Trypsin activates other zymogens:i. Chymotrypsinogen; Proelastase;

Procarboxypeptidaseii. Pancreatitis: tissue damage and pain due to uninhibited trypsin

1. Non-hereditary: Zymogens dont get properly released due topancreatic duct blockage; get activated and overwhelm trypsininhibitors

a. Trypsin inhibitor-trypsin complex is so tight trypsincant digest its peptide inhibitor

2.

Hereditary: Change of a particular amino acid in the trypsinmolecule can disrupt the interaction with its peptide inhibitor (abackup safeguard mechanism in the pancreas)

3. Acute: Pain is centered in the upper middle or upper left part ofthe abdomen. Often temporary

4. Chronic: diminished pancreas function5. Amino acid absorption: uptake into and out of intestinal cells occurs through

transporters

a. Amino acid concentration low: lumen and bloodb. Amino acid concentration high: inside intestinal cells

i. Active transport using Na+ linked systemsspecific for groups of amino acids

1. There are at least 7 different brush borderspecific transport systems

2. E.g. A-system: small neutral amino acids;cysteine transporter

ii. A gradient of sodium (outside high, inside low) iscreated by Na+/K+ ATPase

iii. Transporters in the brush-border membranesimultaneously move Na+ and an amino acidacross the membrane, using the energy of theNa+ gradient to concentrate amino acids withinthe cell

Enters

portal vein

on the way

to the liver

(low aa conc)

(higher aa

conc)

(lower aa conc)


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6. The kidney normally reabsorbs amino acids for re-usea. Is nearly 100% efficientb. 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 tohigh levels of cystine in urineis oxidized leading to kidney stones

iii. Treatment involves making the urine more alkalined. Hartnup disease

i. Defects in a different transporter, results in the abnormal excretion ofseveral neutral amino acids, including tryptophan into the urine; alsodeficient 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 dementiaalthough intermittent and slightly less severein comparison) due to tryptophan deficiency, especially after periodsof poor nutrition

iv. The effects of the disease occur mainly in the brain & skin, even though the underlyingdisease is in the cells of the kidney and intestine; Rash develops on parts of the body exposedto the sun; Mental retardation, short stature, headaches, unsteady gait, and collapsing orfainting are common; Psychiatric problems may also result such as anxiety, rapid moodchanges, delusions, and hallucinations)

v. E.g. Niacin requirements study of 1952: Seven subjects weremaintained 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 machineryneeded for muscle breakdown;muscle contributes more than 60%of the total amino acid pool ofblood)

8. Mechanisms of protein degradation9. Lysosomal system(minor

contribution)a. Cathepsins (lysosomal

proteases) are effective forbacteria, apoptotic

fragments, extracellularproteins (those in LDL), etc

b. Inhibitors of lysosomalfunction (e.g. chloroquine)have little effect on thedegradation of mostintracellular proteins

10.Digestive enzyme turnoverCachexia is the loss of weight, muscle atrophy, fatigue, weakness and appetite insomeone who is not actively trying to lose weight.


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a. The digestive enzymes are themselves being degraded and contribute aminoacids back to the pool (~70 grams/day)

i. About the same level of digestive enzymes are being synthesized11.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 aparticular signal

b. Final product is apolyubiquitinated proteindestined for the proteasome inthe cytoplasm

c. Proteasome componentsi. Regulatory protein:

selects substrates;removed Ub forrecycling; edits wronglytagged proteins

ii. Barrel core: inside,peptides are digested viaproteases to peptides of 7to 10 amino acids

iii. Some of these peptidescan be transportedthrough the ER for presentation to the immune system by MHC Class Imolecules; others are degraded to amino acids by other cytosolicproteases 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 E3enzymes varies among tissues and physiologic states

e. Protein degrade at different rates; in general, proteins should be around for along 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 Signalsi. In the protein sequence; not variable

1. N-end rule(methionine is slowest)2. PEST sequences3. Destruction boxes

ii. External factors; more variable1. Phosphorylation

Proteasome Functions

5 & 6: other non-Ub methosending proteins to th

Proteasome

Step 8 >> Step

Abundantaminopeptidases inside

most cells

Tap: Transporter Associawith Antigen Processing

Important point: Ubiquitin isrecycledin a reaction by the

deubiquitinase (DUB) enzyme


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2. Denaturation/ damage3. Facilitators/ Chaperones; interaction with other proteins

determines the likelihood that a particular protein will beubiquinated (e.g. HPV16E6)

4.

E6 protein of HPV bindsto the tumor suppressorp53 and to E6AP (aparticular E3 with aHECT domain); thisinteraction results in theubiquitination anddegradation of p53 andcontributes to theoncogenicity of the virus

12.Abnormalities in the UPP in diseasea.

Loss of function mutations resulting inincreased stability of substrates

i. E.g. cancer from stabilization of oncogenesb. Gain of function mutations resulting in decreased stability or half-life

i. E.g. cancer from destabilization of tumor suppressors (p53)c. Parkinsons Disease

i. Most cases of Parkinsons diseaseare characterized by accumulation ofubiquitin conjugates (Lewy bodies =protein deposits) in the brain

1. The Lewy bodies cause aloss of neuronal cells & lossof dopamine, a neuraltransmitter

2. The most widely used formof treatment is L-dopa invarious forms.

ii. Several mutations can causeParkinsons

1. Defect in Parkin protein: aubiquitin-conjugating ligase

2. Malfunctioning deubiquitinase13.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 intracellularamino acids (15-30 mM)

c. Alanine and glutamine are the most abundant amino acids in plasma (it'salanine, glutamate, glutamine, and glycine inside cells)

Form complex with target protein

Increase(or decrease) likelihood of ubiquitination

HPV E6 facilitates the degradation of p5

The presence of the viral E6 protein increases ubiquitination

by recruiting E6-AP, a E3 ubiquitin ligase

p53, cell cycle, # of cells

Certain types of HPV are associated with cervical cancer

Several mutations can cause Parkinsons Disea

Usually Lewy Bodie

surviving neuron

Loss of neuronal cells & l

dopamine, a neural trans

d

Parkin is a E3-typeubiquitin ligase

UCH-L1is a

deubiquitinase


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i. These do not reflect their abundance inproteinsit's more their metabolism

d. All 20 amino acids have to be maintained in the bloode. Some Amino Acids are Essential and must be

obtained from degradation of ones own body protein

or the dieti. Amino acids cannot be stored for later use; all20 must be present in approximately correctproportions for protein synthesis

ii. Adults should consume at least 55g proteinper day, otherwise breakdown of body proteinwill occur

f. Kwashiorkorresults from inadequate protein or incomplete protein althoughtotal 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

29. Protein Degradation & Nitrogen

1. N-acetyl glutamate synthetase deficiency (NAGS)a. Tremors, slowness of motion, loss of balance,

stiffnessb. Treatment: Carbaglu pills

2. Nitrogen excretion in urinea. Ammonia (NH4+): normally < 0.1 mM in

blood, a little in urineb. Urea: >90% of N in humansc. Uric acid: a little in urine; is a purine

breakdown product; creatinine is proportional to muscle mass

Arginine Valine Phenylalanine Histidine ThreonineMethionine

Are valuable for his thriving metabolism

IsoleucineLysine 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"

Thr Trp Val Arg His Lys Phe Leu Ile Met

Essential Amino Acids needed for

"Complete Protein

ANIMAL VEGETABLE

10 - 25% protein 1 - 2% protein

Easily digested Incompletely digested

Nearly complete AAs Low in essential AAs

Lysine, methionine, threonine

(legumes & grains are complementary

Chemical Score is high Chemical Score is low

Comparing Protein Sources

Body Protein

220 g/d

Amino Acid Pool

Dietary Protein

70-100g/d

NH3 Carbon ske

Urea glucose, fa

Feces10g/d

Protein Synthesis

Degradation

Digestive Juices

70-100g/d

Synth

Nitr

con

Com


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3. Nitrogen of degraded amino acids shows up mostly as ureaa. Under conditions of constant weight (nitrogen

balance), only the urea level changes as a result ofchanged protein intake

b. No protein reserves created as a result of 'excessive'protein intakec. Only the carbons from excess amino acids liberatedfrom proteins are stored (or burnt to CO2as fuel)

4. Nitrogen balance: urea levels are proportional to proteinload

5. Positive nitrogen balance: increased dietary protein; bodyprotein is increased; the nitrogen is ending up in protein

6. Negative nitrogen balance: loss of protein due to the use ofbody protein

7. The carbons of excess amino acids are used; the nitrogen iswaste

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 urea8. 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; pyruvateii. Corresponding amino acids: glutamate;

asparate; alanineiii. There are no keto-acids for lysine or

threonineb. Enzyme: aminotransferase

i. Coenzyme: PLP = pyridoxal phosphate1. Arises from Vitamin B6

a. B6 deficiency associatedwith dementia due to lackof serotonin productionfrom tryptophan

2. PLP exchanges a keto group formethylenyl amine group

9. The N content of Glutamate is used to make ureaa. Most cells have plenty of transaminases and glutamate dehydrogenaseb. Release of NH4+ in liver can be incorporated in ureac. Urea production is significant only in the liver (multiple enzymes are

required)

Dietary Protein Amino acid pool U

Balance

Normal Adult, not gaining muscle or protein (weight stable)

Body Protein(and digestive enzymes)

Nitrogen Balance

Positive BalanceGrowth, Pregnancy, Muscle Building

Dietary Protein Amino acid pool U

Body Protein (is increasing

Glutamate Dehydrogenase

The reaction catalyzed by glutamate dehydrogenase is key& ATP & GDP

Ammonia is also produced from amides

asparagine and glutamine

Several reactions

produce ammonia; w

do you do with thammonia?

3 ways to dispose o

1. Make glutamat2. Make glutamin

3. Make urea


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d. Glutamate dehydrogenase produces ammoniai. Reversible, regulated step

ii. Forward reaction is fuel-burningiii. Reverse reaction can be used to fix ammoniaiv. Located in mitochondria; there are abundant

glutamate and KG, ATP/ADP transporters10.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 thebrain, muscle, lungs, and adipose cells

i. Glutamine is a major carrier of N in the blood; glutamine levelsincrease after a protein rich meal

ii. Glutamine levels can only rise so much to absorb ammonia: whenblood glutamine plateaus, bloodammonia is excessive (e.g. fatal forinfant born into a family with a

history of hyperammonemia)11.Urea Cyclea. Some urea cycle enzymes increase 10-fold or more after a

protein-rich mealb. The liver has ALL five enzymes & proteins for urea

synthesis (requires other transporters, enzymes to functionfully)

c. 2 steps in mitochondria; 3 in cytosoli. Make NH4+in the mitochondria of liver cells for

incorporation into carbamoyl phosphate (andeventually, urea)

ii. Ornithine enters mitochondria, combines withcarbamoyl phosphate (catalyzed by ornithinetranscarbamoylase), exits as citrulline

iii. Production of carbamoyl phosphate iscontrolled by NAG, which is controlled byarginine

iv. Urea is produced from arginine (catalyzed byarginase)

d. A deficiency in any of the enzymes resultsin elevated ammonia

e. The availability of carbamoyl phosphate iskey; adding arginine usually helps

f. One nitrogen comes directly fromasparate, the other from NH4+

g. Ordinarily Careless CrappersAre Also Frivolous About

Urination (ornithine; carbamoylphosphate; citrulline; aspartate;

argininosuccinate; fumarate;

arginine; urea)

12.Genetic Defects: Urea Cyclea. Features of an inoperative or

stressed cycle:Developmental

Pi

Stryer

2 ATP

Arginase

Ornithine

transcarbamoylase (OTC)

argininosuccinase

Argininosuccinate

synthetase

Carbamoyl-phosphatesynthesis in the

mitochondria is

catalyzed by CPSI

NH4+comes from the glutamate dehydrogenase and the

glutaminase reactions

NAG synthase deficiencyis treated with carbamoy

glutamate

NAG is found only in mitochondriaNAG synthase is activated by arginin

(a feed-forward disposal pathway)

The big picture of Nitrogen Excretion

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


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delays; Mental retardation, seizure; Protein intoleranceb. Diagnosis: Blood/urine amino acid analyses for ammonia (perhaps high

arginine for arginase deficiency or high glutamine levels); Enzyme assayc. Treatment:Restrict protein (Nitrogen production); supplement with arginine;

Increase Nitrogen excretion (using medications that bind amino acids:

benzoate, or phenylacetate orphenylbutyrate)13.The urea cycle interconnects with carbohydrate metabolism: OAA; KG, fumarate

14.Arginine is a useful precursor for many compoundsa. 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 prolinec. Arginine is made from argininosuccinate and the action of argininosuccinase

15.Arginine, ornithine, citrulline, and proline are metabolically linked via a glutamatederivative

30. Amino Acid Breakdown

The Urea cycle interconnects with carbohydrate metabolism

3 major points of connection:

Aspartate/oxaloacetate

Glutamate/ -ketoglurateFumarate/malate


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1. Amino acids released from proteins (mostly muscle) can be used for making glucoseor for fuel

2. Glycogenic: after deamination, the carbon skeletons of the amino acids areintermediates of glycolysis or the TCA cycle and can contribute to the synthesis ofglucose 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 moleculecontributes to acetyl-CoA, another part is converted to a gluconeogenicintermdiate

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 intocarbohydrate because of the irreversibility of pyruvate dehydrogenase

a. Lysine and leucine are metabolized to acetyl-CoA and hence don't makeglucose

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[glycinethreonine], cysteine)

a. serine-pyruvate transaminase to make alanine and hydroxypyruvate in 2 more steps converted toglycerate-2-P and then to pyruvate

b. The details of cysteine & threonine degradation (and tryptophan and histidine) are not examined in thiscourse

c. Dehydratasesacting on amino acids result in the production of differentketoacids

i. The following are glycolytic substrates:

30. Amino Acid Breakdown the

Carbon skeletonsBody Protein

220 g/d

Amino Acid Pool

Synthesis

(non-essential)Dietary Protein

70-100g/d

Conversion to

purines, pyrimidines,

heme, creatine,amines

NH2 Carbon skeleton

NH3 Urea Carbo. Fat

CO2

Feces10g/d

Protein Synthesis

Degradation

Digestive Juices70-100g/d

Fates of the amino acids:

Glycogenicor

Ketogenic

You can't get bloglucose from

leucine and lysiSome amino acidshave multiple entrypoints (Threonine,

Tyrosine in 2 parts)


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ii. Serinepyruvateglucoseiii. Threonine-ketobutyrateglucose

5. -ketoglutaratepathwaya. Proline, glutamine, arginine, glutamate are broken down to KG

6. Succinyl-CoApathwaya. Methionine, isoleucine, valine, threonine

b. Transamination to form the corresponding -ketoacidsc. Oxidative decarboxylation by a dehydrogenase complex: the branched chain

2-keto acid dehydrogenase(BCKADH), containing thiamine pyrophosphate,lipoate, and FAD

i. Mechanism similar to pyruvate dehydrogenaseii. When BCKADH is missing, the 3 branched chain keto acids

(isoleucine, valine, leucine) as well as -ketobutyrate (frommethionine and threonine metobilism) accumulate

1. By reversal of the aminotransferase reaction, the blood levelsof the branched chain amino acids also rise

2. -Ketoglutarate Pathway Arginine, Proline, Histidine, Glutamine, GlutamateArginine

Ornithine

Proline

Glutamicg -semialdehyde

urea

Glu

-Kg

Glutamate -Ketoglutarate

Glutamine

Histidine

NH3

NAD+ NADH + NH3

-- -

ProlineProlinedehydrogenase

g

3. Succinyl CoA Pathway Threonine, Methionine, Isoleucine, Valine

IsoleucineMethionine

Threonine

Valine

Propionyl CoA

Succinyl CoA

-keto- -methylvalerate -ketoiso alerate -ketobutyrate

AT AT

NH3DH*DH*DH*

AT - amino transferasesDH* - branched-chain ketoacid dehydrogenase(see section on maple syrup urine disease formore details)

Succinate

Note: -ketobutyrate and other a-ketoacids are metabolized in reactions similar to pyruvatedehydrogenase and require thiamine, lipoate, FAD


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2. Maple Syrup Urine Disease: individuals that lack the enzymecomplex and build up keto acids, resulting in urine that smellsof maple syrup

a. Treatment includes severe restriction in the intake ofbranched chain amino acids

iii.

The propionyl-CoA derived from isoleucine, threonine, valine, andmethionine are metabolized to succinyl-CoA1. Requires biotin and vitamin B12

a. Vitamin B12 is required for metabolism of valine,isoleucine, & methionine

b. 3 to 40% of older adults have various degrees of B12 deficiencyc. Symptoms range from lethargy and weight loss to dementia

2. Adenosylcobalamineis formed from Vitamin B12a. adenosyl form of Vitamin B12 is needed for the mutase

reactionb. 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 methylmalonyl-

CoA to succinyl-CoAa. Several possible deficiencies: dietary intake of VitB12,

lack of intrinsic factor, or an inability to convert to theadenosyl form, or deficient enzyme

b. Results in hematopoietic and neurological disordersc. Treatment: administer large doses of

Adenosylcobalamine4. Mutase reaction defect; methylmalonate appears in the urine

a. Underlying problem can be complex: missing enzymeor vitamin B12, problem with its absorption, or itsprocessing

b. Unresponsive to Adenosylcobalamine dosing7. OxaloacetatePathway

a. Asparagine is degraded to asparate (via asparaginase) which is thendegraded to OAA (via asparate aminotransferase)

i. The NH4+of aspartate is passed on to -ketoglutarate to makeglutamate in a transamination reaction

8. Fumaratepathwaya. Phenylalaninetyrosine

fumarate & acetoacetate

b. The products are glucogenic &ketogenicc. Phenylalanine is an essential

amino acid; when deficient, themetabolites made from it arealso deficient

d. Phenylketonuria(PKU)

Dihydrobiopterin reductase

regenerates tetrahydrobiopterin

(Tetrahydrobiopterin is synthesized from GTP)Tyrosine becomes an essential

in individuals with PKU

*possible mutation

*possible mutation


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i. Build-up of phenylpyruvic acid; associated with mental retardationii. Different forms; can be deficient in:

1. Phenylalanine hydroxylasein the liver involved in convertingdietary phenylalanine to tyrosine

2. The ability to make tetrahydrobiopterin(a necessarycofactor)a. 50% of PKU patients are responsive to tetrahydrobiopterin replacement

iii. Guthrie testan assay for the presence of high levels of phenylalanineiv. High concentrations of phenylalanine cause brain damage

1. High phenylalanine levels prevents other amino acids beingtransported into brain, perhaps also interferes with protein andneurotransmitter (serotonin) production

v. With phenylalanine metabolism blocked other minor compounds such as phenylpyruvate areproduced

1. Urine is typically musty2. Mothers with PKU must control phenylalanine levels in diet when pregnant

vi. Treatment:1. Protein restricted diet, low phenylalanine2. Supplement for Tyrosine, perhaps supplement a co-enzyme3. Monitor serum levels of phenylalanine closely

e. Alkaptonuriais characterized by dark urinei. Due to a deficiency in homogentisic acid oxidase

ii. Later symptoms: arthritis, due to homogentisic forming crystals in thespine leading to cartilage damage and osteoarthritis

9. Inborn errors of amino acid metabolism are mainly pediatric diseasesa. 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 urineb. 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 Defective Process Defective Enzyme Symptoms and

effectsAlkaptonuria Tyrosine

degradationHomogentisate 1,2-

dioxygenaseDark pigment in

urine; late-developing arthritis

Argininemia Urea synthesis Arginase Mental retardation

Argininosuccinic

academia

Urea synthesis Argininosuccinatelyase

Vomiting,convulsions

Carbamoyl

phosphate

synthetase I

deficiency

Urea synthesis Carbamoylphosphate

synthetase I

Lethargy,convulsions, early

death

Maple Syrup urine

disease (branched-

chain ketoaciduria)

Isoleucine, leucine,and valine

degradation

Branched-chain

keto acid

dehydrogenasecomplex

Vomiting,convulsions, mental

retardation, earlydeath

Methylmalonic

academia

Conversion ofpropionyl-CoA to

succinyl-CoA

Methmalonyl-CoA

mutase

Vomiting,convulsions, mental

retardation, early


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death

Phenylketonuria

(PKU)

Conversion ofphenyl-alanine to

tyrosine

Phenylalanine

hydroxylase

Neonatal vomiting,mental retardation

31. Synthesis Using Amino Acids

1. Different organs use different amino acids differentlya. Niacinb. Nitrous oxidec. 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/ creatinine2. 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 ofPLP in the decarboxylase reaction


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

i. Made from the iodination and covalent bonding of two tyrosineresidues on thyroglobulin

ii. Thyroxine is cleaved from thyroglobulin and secreted from the thyroidiii. Graves Disease: patients develop antibodies against a receptor that

results in over-stimulated thyroglobulin1. 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 tyrosinaseii. Albinism: patients lack tyrosinase and are unable to make melanin,

leading to sun sensitivity and improper eye development3. Biosynthesis using Folate: Synthesis of Glycine, Serine, Cysteine & Methionine

a. The synthesis of: Glycine & Serine & Cysteine (& Methionine) require 1-carbon transfers

b. Several coenzymes are neededc. Folates provide one-carbon fragments for amino acid &protein metabolism

i. Are also involved in synthesis of purines &pyrimidines, nucleic acids, phospholipids, andregeneration of methyl methyl groups used fortransamination by methionine

ii. Low folates can block hematopoiesis, resulting in release of immatureRBCs and megaloblastic anemia

d. Antifolates: folate metabolism is a target for antibacterial agents because of its central role in synthesisi. 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 rapidlygrowing cancer cells by interfering with the synthesis of pyrimidines

e. Tetrahydrofolate(THF) is derived from folic acidi. All but one of the glutamylresidues are removed in theintestinal mucosa, then freefolic acid is reduced bydihydrofolate reductase toTHF

ii. THF is transported in plasmaas the methyl derivative

iii. Inside cells, THF isreconverted to the

polyglutamyl forms (whichare most effective for one-carbon transfers)

iv. Methyl groups canbe carried by N5and

N10 on the

methylpterin ringv. By carrying methyl

groups (in differentformats) at the N5and N10 atoms,

Cofactor Group TransferredBiotin, PLP CO2

Tetrahydrofolate HC=O (formyl or formin-CH2OH (methylene)

-CH3 (methyl, for 1 rea

S-adenosyl methionine-CH3 (methyl, preferred

Most of the forms of folatecan readily interconvertFolate

Dihydrofolate

reductase

These formsof THF are

known as the

one-carbon

pool

The

form

mos

abun

in bl

plas

is fo

in an

irrev

reac


16/39

16

folates can carryout essential methylation reactionsvi. Requirement for folates in nucleic acid synthesis is very important for rapidly growing tissues

vii. Folates are found in green leafy vegetables, beans, lentils, some fruitsviii. 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 andtetrahydro forms of folate (via seri ne hydroxymethyl-transferase)

1. The tetrahydrofolate(H4folate) is converted to themethylene formas glycine is made from serine (and vice versa)

2. Serine is made from the glycolytic intermediate 3-phosphoglyceratex. One carbon fragments can also be derived from tryptophan and histidine

f. S-adenosyl methionine(SAM): can also transfer methyl groups in addition toTHF

i. Synthesized from methionine via methionine adenosyltransferaseii. Can be used in synthesis of epinephrinefrom norepinephrine(which

is made from dopamine)

iii.

The production ofcreatinerequires SAM1. Synthesized from glycine, arginine, and methionine2. Creatine phosphateis a high-energy compound that acts as a

reserve energy supply in muscle by phosphorylating ADP toATP (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 toresistance training.

iv. Folate can be used to regenerate methionine from S-adenosylhomocysteine (SAH)

1. Methionine synthaseuses methylTHF to methylate VitaminB12to make CH3-B12 (aka methylcobalamin cofactor)

a. The enzyme uses CH3-B12 to remethylatehomocysteine to methionine

2. Methylmalonyl mutase& methi onine synthaseare the only 2enzymes known that require cofactors derived from VitaminB12

a. The Folate Trap: The only way in which the THF inmethyl-THF can be returned to the THF pool is via theB12-depedent methionine synthase reaction since themethylenetetrahydrofolate reductase (MTFR) reaction(leading to formation of methyl-THF) is essentially

irreversibleb. Vitamin B12deficiencies lead to:

i. Defects in methionine synthase reactionsii. Build-up of methyl-THF

iii. Decreased levels of available THFiv. A secondary deficiency in folatev. Deficiencies of functional folate can arise from a lack of

dietary folate or vitamin B12


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17

3. Methionine can also be regenerated from homocysteine usingcholine

4. Because homocysteine is used for other reactions, methioninecan be in short supply

g. Degradation of homocysteinei. 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 makecysteine

1. Cysteine is derived from methionine and serine2. Cystathionine synthetaserequired PLP3. If methionine is deficient, cysteine is also deficient4. Degradation of methionine forms -ketobutyrate (succinylCoA)

5. Taurine(2-aminoethanesulfonic acid) is derived from cysteinea. 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 synthetaseii. 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 bonedevelopment); vision problems; psychiatric disturbances; before the age of30, almost one fourth of untreated patients die as a result of thrombotic complications

v. General treatment: restrict protein, supplement with vitamin-derivedcofactors, 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 synthesisb. 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 liveri. Branched chain amino acids(BCAA) isoleucine, leucine, and valine

are preferentially metabolized as fuel in muscle(where theirtransaminases are primarily localized)

1. Carbon skeletons are oxidized to give ATP2. NH3ends up as Glutamine or Alanine and leaves the muscle

a. Alanineis transaminated to pyruvate in the liver andserves as a substrate for gluconeogenesis

i. The resulting glucose can be used in the Cahillcycle(to regenerate alanine), which provides a


18/39


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19

pyrophosphate (PRPP) [via PRPP synthetase]2. Next is replacement of the pyrophosphate on PRPP with the

amide group of glutamine to form 5-phosphoribosylaminea. Committed stepin purine nucleotide biosynthesis; is

when the glycosidic bond is formed as the first atom of

the purine ring is incorporatedb. Enzyme: glutamine phosphori bosyl pyrophosphateamidotransferase (PRPP

amidotransferase)i. Is inhibited by the end

products of purinebiosynthesis

ii. Formation of inosine monophosphate1. 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 produced3. Four ATP molecules are used

iii. Conversion of IMP to AMP and GMP1. Inosinic acid serves as a precursor for

both AMP and GMP2. Synthesis of AMP (from IMP) requires GTP; synthesis of

GMP requires ATP thus, an abundance of one purinenucleoside triphosphate ensures the production of the second;this arrangement helps to insure equal levels of purines

iv. Conversion of monophosphates to diphosphates and triphosphates1. Conversion of Monophosphates to Diphosphates

a. This conversion is catalyzed by kinases that are base-specificbut not sugar-specific (e.g. GMP kinase)

2. Conversion of Diphosphates to Triphosphatesa. Nucleoside diphosphates and triphosphates are

interconverted by nucleoside diphosphate kinaseb. Enzyme has broad specificity

v. Activation of Antiviral Nucleoside Analogues1. Many nucleotide analogues require the

formation of their corresponding 5triphosphates (TP)

2. For example, in order to function AZT mustbe converted to AZT triphosphate

3. AZT triphosphate is formed by the sequentialaction of thymine kinase(nucleoside),thymidylate kinaseand 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 ofnucleic acids)

ii. Two salvage methods:

Formation of Inosinate


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20

iii. Direct conversion of a base into a ribonucleotides1. Hypoxanth ine- guanine phosphori bosyl transferase(HGPRT)

a. Guanine (Base) + PRPPGMP + PPib. Hypoxanthine (Base) + PRPPIMP + PPi

2. Adenine phosphori bosyltr ansferase(APRT)a.

Adenine (Base) + PRPP

AMP + PPiiv. Reversible conversion of bases to nucleosides and then nucleotides

1. Pur ine nucleoside phosphorylasea. Nucleoside N + Pibase + ribose-1-phosphate

b. *Rarely used to salvage a base; the predominant direction of the reactioncatalyzed by this enzyme is derivative

2. Nucleoside kinasea. Nucleoside N + ATPNMP + ADP

4. Catabolismof purine nucleotidesa. Conversion of nucleotides to nucleosides

i. AMPAdenosine (nucleoside) + Pi (via hydrolysis byphosphatases)

b.Adenosine deaminase reactioni. 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 tosupport synthesis via the salvage pathway

d. Uric Acid Formationi. Hypoxyanthine: primary breakdown product of AMP via adenosine

and inosine; is oxidized to xanthine, then touric acid (via xanth ine oxidasein both steps)

ii. Guanine (breakdown product of GMP) isconverted to xanthine by guanine deaminase

5. Regulation of purine biosynthesisa. De novo purine biosynthesis regulation occurs at:i. PRPP synthetasereaction

1. Activity depends on intracellularconcentrations of several end products(AMP, GMP, ADP, etc) of whichPRPP is a substrate

ii. Amidophosphoribosyltransferasereaction1. Salvage pathways generate AMP and GMP through

APRT and GHPRT phosphoribosyltransferasereactions

2. Salvage pathways shut off the denovo pathwaysat the PRPPamidotransferase step

3. AMP & GMP are feedbackinhibitors

a. The amidotransferase has separateallosteric sites for these two nucleotides

4. PRPP is consumed during the formation ofpurinesin the salvage pathway, which decreasesthe rate of formation of 5-phosphoribosylamine

Salvage shuts off de novo pathway

V

PRPP Conc.

phosphoribosy

(salva

amidotran

Control Pathways for Purine Biosynthesis


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21

iii. Branch point in the formation of AMP and GMP from IMP1. AMP & GMP negatively regulate their respective formation

from IMP6. Disorders of purine nucleotide metabolism

a. Gout: elevated uric acid levelsin the blood, due to a variety of metabolicabnormalitiesi. 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 crystalsare deposited in the cartilage of joints resulting in painand disability (also deposited in the kidney producing renal damage)

iii. Can be due to:1. Overproduction of purines through defective control mechanisms2. 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 salvagepathways; PRPP accumulates and causes activation ofde novo purine biosynthesis

b. The lower IMP & GMP levels result in reducedfeedback inhibition of the de novo pathway

2. PRPP synthetaseenzyme (increased activity)a. Is less susceptible to feedback inhibition by purine nucleotidesb. The increased enzyme activity leads to

overproduction of PRPP, and thus activation of the denovo pathway

3. Glucose-6-phosphataseenzymea. Enzyme deficiency leads to increased utilization of

the pentose-phosphate pathway, and consequently, toexcessive production of ribose-5-phosphate, the

immediate precursor of PRPPv. Allopurinol: lowers uric acid levels in the blood

1. Analogue of hypoxanthine2. 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 syndromei. Infants with this disease lack a functional HGPRT

ii. Marked increase in the rate of purine biosynthesis by the de novopathway

iii. Symptoms include self-mutilation, mental illness and gout likesymptoms owing to elevated uric acid in serumiv. The mechanism by which the deficiency of HGPRT causes central nervous system disorders

remains unknown

1. However, in normal subjects, HGPRT activity is highest inthe brain, suggesting the importance of the purine salvagepathway in this tissue

v. Allopurinol is used to treat the Gout but there is currently no cure for the neurologicalproblems


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22

c. Adenosine deaminase deficiencyi. Causes one form of severe combined immunodeficiency(SCID):

patients lack both B and T lymphocyte functions and are usually deadfrom 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

dATP7. Inhibitors of purine biosynthesis

a. Sulfonamidesi. 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(acomponent of folic acid) and inhibit synthesis of folate in bacteria

1. Folate is required in de novo biosynthesis (and also for thymidylate and methioninebiosynthesis)

iii. Blocks the formation of an essential bacterial compound and thus,their growth

b. 6-Mercaptopurinei. Functions as an analogue of hypoxanthine1. Is converted to a nucleotide by HGPRT, which draws some of

the PRPP away from nucleotide biosynthesis pathways2. 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, iscompetitive inhibitor of IMP pathways for AMP & GMPbiosynthesis)

iii. Used in the treatment of acute leukemia(antitumor drug)

33. Pyrimidine (T/C) Metabolism

1. Synthesis of pyrimidinesa. Pyrimidine skeleton is assembled first and is subsequently

attached to PRPPb. A major regulatory step is formation of carbomyl

phosphatec. De Novo Synthesis:d. Step I: Carbamoyl phosphate synthase I I

i. Takes place in the cytoplasm and IS NOT part ofthe urea cycle (urea cycle takes place in liver mitochondria and is catalyzed by a different

enzyme)ii. Glutamine + 2 ATP + HCO3-Carbamoyl phosphate + 2 ADP +Glutamate

e. Step II: Aspartate Transcarbamoylasei. Carbamoyl phosphate condenses with aspartate

f. Step III: Synthesis Of Orotatei. The ring is closed in the dihydroorotasereaction


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ii. Dihydroorotase dehydrogenasemakes orotate, the immediateprecursor of all pyrimidines

iii. All three steps take place on one protein with multiple enzymaticactivities

g. Step IV: Synthesis of UMPi.

Orotate condenses withPRPP to form orodylateii. UMP is next generated

by a decarboxylationreaction

h. Step V: UTP Formationi. Ur idylate kinase: UMP + ATPUDP + ADP

ii. Nucleoside diphosphate kinase: UDP + ATPUTP + ADPi. 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 phosphorylase2. Pyrimidine catabolism

a. In contrast to purine catabolism, pyrimidine catabolism (which occurs mainlyin the liver) yields soluble end products

b. Nucleotides are first converted to nucleosides by phosphatasesc. Cytidine is converted to uridine by cytosine deaminased. Uridine and thymidine (nucleosides) are converted to free bases by pyrimidine

nucleoside phosphorylasei. Catabolism of uracil and thymine (bases) proceed in parallel steps;

catalyzed by the same enzymesii. Large numbers of cells are killed, and DNA degraded, in cancer

patients undergoing chemotherapyiii. It is possible to estimate the turnover of DNA by measuring b-

aminoisobutyrate(end product of thymine pyrimidine catabolism)3. Synthesis of Deoxyribonucleotides: Ribonucleotide Reductase

a. All four ribnucleoside diphosphates (ADP, GDP, CDP, UDP) can beconverted to the deoxyribonucleoside by ri bonucleotide reductase

b. The electron donor for this reaction is NADPH viaoxidation of the proteinthioredoxin

i. The sulfhydryl groups are regenerated by a reaction with NADPHcatalyzed by thioredoxin reductase

ii. Hydroxyurea(an antineoplastic agent and inhibitor of DNAsynthesis) inactivates the enzymeribonucleotides reductase


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4. Control mechanisms for nucleotide formationa. Mechanism of Ribonucleotide Reductase

i. Two sulfhydryl groups onribonucleotide reductasereduce the ribonucleoside

to the deoxyribonucleosideii. Thioredoxin sulfhydrylsregenerate the oxidizedsulfhydryls onribonucleotide reductase

iii. Thioredoxin reductaseregenerates the sulfhydryls on thioredoxinusing NADPH

b. Regulation of de novopyrimidine metabolismi. In mammalian cells, regulation occurs primarily at the level of

carbamoyl phosphate synthetase IIii. Next level of regulation is at the level of OMP-

decarboxylase1. UMP is an inhibitor ofOMP-decarboxylasec. Control of biosynthesis of dNTPs

i. The reduction of ribonucleotide diphosphates iscontrolled by allosteric interactions

1. E.gbinding of dATP to allosteric sitesonri bonucleotide reductaserenders theenzyme inactive

2. Alternatively,binding of ATP activates theenzyme

ii. Regulation permits a fine adjustment of the dNTPpools required for DNA synthesis

5. Pyrimidine analoguesa. Synthesis of Thymidine

i. TMP is formed by the methylation of dUMPii. Reaction catalyzed by thymidylate synthase

iii. N5, N10-methylene tetrahydrofolate is the one-carbon donor


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b. Nucleotide metabolism as targets forchemotherapy

i. Thymidylate synthaseinhibitor: Fluorouracilends up being covalently

attached to the enzyme,essentially killing the enzyme(suicide inhibitor)

ii. Dihydrofolate reductaseinhibitor: Aminopterinand

Methotrexate1. Methotrexate is a

Folate Analogue and aCompetitive Inhibitor of Dihydrofolate Reductase

6. Coordination of purine and pyrimidine nucleotide biosynthesisa. PRPP activates carbamoyl phosphate synthetase IIand is a rate limiting

substrate for orotate phosphoribosyltransferase.b. Recall that PRPP activates amidophosphoribosal-transferase and that PRPP isthe substrate for HGPRT and APRT

34. Integration of Metabolism

1. Food and fuel (or fat)a. Storage

i. 400 gm (1700 cal) Glycogenii. 6,200 gm (24,000 cal) Protein

iii. 15,000gm (138,800 cal) and up Triglyceridesb. Energy: Variable demand

i. 1,400 (couch potato)ii. 3450 calories (runner)

iii. 9,000 (severe burns; infection) C/day2. Fuel transport between organs

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

What happens when you eat?

Food Circulation


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b. Liver produces ketone bodies or FA for muscle heart and brainc. Lactate from muscle or RBS comes back to liver to make glucosed. Liver maintains the glucose levels; can use fatty acids and glycerol to make

glucosee. Type I diabetes & starvation: liver can generate ketone bodies (source of

energy due to excess acetyl CoA)3. Different organs work differentlyTissue Storage Fuel Preferred Fuel Exported Fuel

Liver GlycogenTriacylglycerol (TG)

GlucoseFatty acids (FA)

Amino Acids

GlucoseFA

Ketones

Skeletal Muscle

(resting)

Glycogen FA

Skeletal Muscle

(working)

Glucose AlanineLactate

Adipose TG FA FA (heart)Glycerol (liver)

Heart FA

Brain Glucose(Ketones in starvation)

4. Links between metabolism of carbohydrates,fats and proteins

5. Regulating individual steps in metabolism

Carbohydrate Fat

Glucose-6-P NADPH

DHAP Glycerol-3-PTG

PL

Pyruvate Acetyl CoA

Fatty acids

Ketone bodies

Cholesterol

TG

PL

Fat Carbohydrate

TG Glycerol-3-P

Glucose

Carbohydrate Protein

GLU-6-P

3-P-glycerate Ser, Gly, Cys

-ketoglutarate Glu, Gln, Pro

Oxaloacetate Asp, Asn

Pyruvate Ala

His

Arg

ProGln

Glu -ketoglutara

Protein Carbohydrate

Trp Ala

Gly, Ser

Cys

Pyruvate

Ileu

Thr

Met

Val

Succinyl CoA

Phe FumarateTyr

Asp Oxaloacetate


27/39

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a. Allosteric inhibition or stimulationi. Rapid response; results in marked changed in concentration in enzyme

activity may result from only a moderate change in its regulatorsconcentration

Allosteric activation in the well-fed state

Enzyme + Effector Effect Activated Pathway

Pyruvate Kinase F1,6BP & F2,6BP Provides pyruvate for fatsynthesis

Glycolysis

Acetyl CoA carboxylase Citrate Stimulation of fatsynthesis

FA Synthesis

Pyruvate Dehydrogenase Pyruvate Provides acetyl CoA forfat synthesis

FA synthesis

Glycogen synthetase Glucose-6-P Increases storage ofcarbohydrate as glycogen

Glycogen synthesis

Phosphofructokinase F2,6BP Provides pyruvate for fatsynthesis

FA synthesis

Allosteric inhibition in the well-fed state

Enzyme - Effector Effect Inhibited Pathway

Glycogen phosphorylase GlucoseDecreases glycogen

breakdownGlycogen

breakdown

Carnitine acyl

transferase-1Malonyl CoA Decreases FA breakdown FA breakdown

Phosphofructokinase Cytoplasmic citrate

Fructose 1,6

BPhosphataseF2,6BP Gluconeogenesis

EXAMPLES OF ALLOSTERIC REGULATIONAll 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 youcan guess what the effect should be based ocommon sense

Citratein excess says that you have plentifCHO: would like to store energy

Malonyl CoA says you are making fat: donwant to break it down so you block transpo

G6Psays that you have plentiful CHO andwould like to store it

Same token, plenty of glucose means youdont want to make more by breaking down

glycogen


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b. Regulation of the amount of enzymei. Inhibition or stimulation of gene transcription

ii. Protein degradation or stabilizationiii. High protein:

1.

Induction of enzymes needed for nitrogen metabolisma. Ornithine carbamoyl transferaseb. Argininosuccinate synthetasec. Argininosuccinase

2. AA catabolism/ gluconeogenica. Serine dehydrataseb. Tyrosine aminotransferasec. Alanine aminotransferased. Cystathionasee. Glutaminasef. Ornithine aminotransferase

iv.

High carbohydrate:1. Glucose uptakea. Amylase(for breakdown)b. Glucokinase(to fix glucose for glycogen synthesis)

2. PPPa. Glucose-6-phosphate dehydrogenaseb. 6 phosphogluconate dehydrogenase

3. Fat biosynthesis(to store excess)a. Acetyl CoA carboxylaseb. Fatty acid synthetasec. NADP-malate dehydrogenased. 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 productiona. Glucose-6-phosphataseb. Serine dehydratase (pyr)c. Ornithine aminotransferase (proline)d. Tyrosine aminotransferasee. Fructose bisphosphatase


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Induced enzymes in well-fed state

Glucokinase Glucose uptake for storage

G6P dehydrogenase

6-phosphogluconate dehydrogenase

PPP provides NADPH for fat synthesis

Pyruvate kinase Glycolysis provides pyruvate for fatsynthesis

NADPH- linked malate dehydrogenase Provided NADPH for fat synthesis

Citrate lyase Provides cytoplasmic acetyl CoA for fatsynthesis

Acetyl CoA carboxylaseFatty acid synthase

Increases FA synthesis

HMG CoA reductase Increases cholesterol synthesis

Induced enzymes in starvation state

Glucose-6-phosphatase Increases glucose relative to blood

Pyruvate carboxylase

PEP carboxykinase

Various amino acid transaminases

Increases gluconeogenesis

c. Covalent modificationi. Inhibition or activation by phosphorylation

1. Phosphorylation can change Km, Vm or localization2. Balances between gluconeogenesis and glycolysis

ii. Dephosphorylation: an insulineffect in the well-fed state (a statewhere you need to absorb glucose)

1. Enzymes activated by dephosphorylation are for:a. Glycogen storage


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b. FA synthesisc. Cholesterol/ TAG synthesis

2. Enzymes inhibited by dephosphorylation are for:a. Glycogen breakdownb. Gluconeogenesis

iii.

Phosphorylation of Hormone-Sensitive Lipase in presence of glucagon1. Allows triglycerides to be broken down to free fatty acids andglycerol

2. Insulin leads to HSLs dephosphorylation and inhibition of thebreakdown of triglycerols & fatty acids

d. Compartmental separation

6. Hormonal control of metabolism

a. Mechanisms of hormone actioni. G-Protein/ Cyclic AMP systems

1. Hormones: glucagon, epinephrine2. All Receptors That Activate cAMP Have

Similar Overall Structurea. They all span the membrane 7 timesb. Highly hydrophobic transmembrane

region

GKMITO Fatty-Acyl CoA

Muscle Regulation of Fatty Acid OxidationVia Compartmentalization

CPT

-I

Malonyl CoA

Acetyl CoA

Acetyl CoA

AMP

AMP-PKACC-2

MDCFatty Acid Oxid

LOW BLOOD GLUCOSE

Pancreas

ACTH

Adrenal

Pituitary

Autonomic NervousSystem

Hypothalamic Reg Ctr

Cortisol Epinephrine Norepinephrine Glucagon

Glycogenolysis

Gluconeogenesis

0

++

+++

0

+++

0

++

++

Adenylate cyclase (cAMP) Cascade

Receptor

G protein

Adenylate cyclase

cAMP

Protein Kinase A

Phosphorylation of enzymes,transcription factors, et cet.


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c. All have the same intracellular domains (same G-protein) but different extracellular domains to providespecificity

3. Mechanism:4. Hetero-trimer inhibitory subunits and GTP/GDP binding

subunits5. Receptor (Guanine nucleotide exchange factor, GEF)causes dissociation of subunits and GDP release allowingGTP to take its place

6. GS- then activates adenylate cyclase7. GS- then hydrolyzes bound GTP to GDP with the help of1. GTPase activating proteins (GAPs) shutting of the process2. GDP Gs then binds again

3. Adenylate Cyclase Synthesizes cAMP (and Phosphodiesterasedegradation of cAMP)

a. Caffeine inhibits the cAMP phosphodiesterase enzyme4.

cAMP activates Protein Kinase Aa. Promotes dissociation of regulatory (R) subunits and

liberation of Catalytic (Cat) subunitb. PKA regulates both glycogen breakdown and synthesis

ii. Tyrosine KinaseInsulinreceptor sets off a signaling cascadeiii. Steroid hormones(Cortisol) bind in the cytoplasm and then move

into the nucleus1. Binds intracellular receptorsand promotes glucose synthesis2. Direct transcriptional activators

7. Starvationa. 1stPhase: well-fed phasethat lasts 4 hours while exogenous fuel is supplying

and maintaining blood glucose levelsi. Gluconeogenesis starts 4-6 hours after food and becomes fully

activated as glycogen is depletedii. 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

Glucagon

R

G-GTP Adenylate

cyclase

Protein kinase A

Cat

Glycogen

synthase

P-Glycogen

synthase

Phosphorylase

kinase

P-Phosphorylase

kinase

P-PhosphorylasePhosphorylase

+ Reg-cAmp

More glycogen breakdown Less glycogen synthesis

Protein kinase A

Cat-Reg

PKA Regulates Both Glycogen Breakdown and Synthesis


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c. 3rd: Early starvation(16- 48 hours)i. Gluconeogenesis from AA:

thus protein breakdown beginsincrease in urea excretion

ii. Glucoconeogenesis is elevatediii.

Triacylglycerols produce FAsfor energy; glycerol foradditional glucose production

iv. Muscle decreases use ofglucose, increases use of FA

1. Muscle glycogen notuseful for anything butmuscle

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 brain1.

Decrease in gluconeogenesisiii. 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 isused mainly by RBCs and the brain

iii. Total amount of nitrogen excreted as urea decreases dramatically tosave protein.

iv. Urinary ammonia initially increases to conserve cations to preventketone body excretion

v. Blood metabolite levels:1. Increase in:

a. Ketone bodiesb. Free fatty acids

2. Decrease in:a. Glucoseb. Urinary nitrogen

3. Urinary ammonia initially increases and then decreases4. Urea increases due to protein breakdown to synthesize glucose,

and then decreases later on to preserve glucose (protein)

Stages of Starvation

Origin ofBlood

Glucose

TissuesUsing

Glucose

Exogenous

All

GlycogenHepatic

gluconeo-

genesis

All except liver.Muscle and fat

tissue at

decreased rate

Hepaticgluconeo-

genesis

Glycogen

All except liver.Muscle and fat

tissue at rate

between III and

IV

Hepatic andrenal gluco-

neogenesis

Brain, RBC,renal medulla

Small amt by

muscle

Hepatrenal

neoge

Brain decre

rate, R

renal

GLUCOSE LACTATE

ATP + CO2

FUEL METABOLISM DURING EARLY STARVATION

(16-32 HRS)

TISSUES BLOOD LIVER

ATP + CO2

STORED FUELS

Triglycerides

(160 g/day)

Protein (75 g/day)

KETONE BODIES

FATTY ACIDS

GLYCEROL

AMINO ACIDS

LACTATE

GLUCOSE

KETONE BODIES

Ac-CoA ATP + CO2

GLUCOSE

FUEL METABOLISM DURING MIDDLE AND LATESTARVATION

TISSUES BLOOD LIVER

ATP + CO2

STORED FUELS

Triglycerides

(160 gday)

Protein (20 g/day)

KETONE BODIES

FATTY ACIDS

GLYCEROL

AMINO ACIDS

GLUCOSE

KETONE BODIES

Ac-CoA ATP + C

GLUCOSE

GLUCOSE LACTATE

ATP + CO2

URINE

LACTATE


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f. Three factors keep blood glucose constant:i. Mobilization of glycogen; gluconeogenesis

ii. Release of free fatty acids1. 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 l ipaseandaided by catecholamines, epinephrine and especiallynorepinephrine

a. In fasting li poprotein l ipaseactivity is low, socirculating TAG not available to make fat

3. Fatty acids are released into the blood; transported bound toalbumin

iii. Switch of muscle and liver to using free FA as fuel

EXTRA SLIDES:

A. After a meal

B. Afterovernight fast

Glucose metabolismin liver

Fuel Flow During an Overnight Fast

Interplay of Urea cycle and gluconeogenesisThe relationship of aa catabolism to gluconeogenesis.

Urea Cycle: Get OAA, fumarate for gluconeogenesis

Interplay of Urea Cycle and Gluconeogenesis


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35. Diabetes

1. Diabetes as a Disease

Fate of Glucose in the Cell

PPP

Ketone BodiesIn case of excess acetyl CoA in starvation, (i.e no OAA for TCA

cycle due to glucose depletion. Then ketone bodies are produced

Fatty Acid Biosynthesis

TCA

Fate of Acetyl CoA


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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: Twicethat of non-diabetics at anyage

v. Total Cost (direct andindirect): $174 billion/yr

vi. Medical Expenses 2.3Xhigher than non-diabetics

b. Diabetes = Starvation in thepresence of plenty

c. Type 1 (5-10%): loss of -cells

i. Little or nocirculating insulin

ii. Normal response toadministration of insulin

iii. Often present with ketoacidosisd. Type 2 (~90%): insulin resistance/cell failure

i. Older onsetii. Cells don't respond to insulin

iii. Generally don't show ketoacidosis2. Insulin Synthesis and Secretion

a. Insulin is produced by processing of precursors (preproinsulin and proinsulin)b. Insulin production and secretion is controlled by:

i. Glucose1. High GlucoseG6PATP closes K channelCa secretioninsulin secretion

2. Insulin Secretagoguesblock potassium channels causingdepolarization

a. Close the ATP-sensitive K+ channel leading to a rise inintracellular calcium, increased fusion of insulingranulae with the cell membrane, and thereforeincreased secretion of (pro)insulin

b. Sulfonylureas; Meglitinide3. Metforminreduces hepatic glucose output

a. Inhibits gluconeogenesis (decreases BGL)ii. Incretins: hormones produced in the digestive tract1. Increases insulin release when orally-ingested glucose levels

are normal or elevateda. Dont act when glucose levels are low

2. GIP = glucose-dependent insulinotropic peptide3. GLP = glucagon-like peptide

a. Acts on secretion via ion channels

Characteristics of the Two Major Types of Diabetes

(other: Gestational,

Maturity Onset Diabetes of the Young (MODY)

beta-cell dysfunction)


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b. Blocking this breakdown enhances insulin secretion4. Exenatide: GLP1/GIP analogue5. Sitagliptin (Januvia): DPPIV protease inhibitor that stabilizes

GLP1/GIP proteina. Blocks the degradation of incretins

3.

Insulin Signalinga. Tyrosine kinase pathwayi. Insulin establishes a cascade with a lipid second messenger

b. Insulin induces glucose uptake by inducing glucose transporters in fat andmuscle

i. Induces Glut4 activity and localization

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

4. Insulin and Metabolisma. After eating:

i. Glucose level goes upii. Insulin level also goes up because glucose stimulates beta cells to

secrete insulin1. Much less increase in insulin after a protein rich meal

iii. Glucagon goes down after a meal balanced with CHO (glucagon isproduced by alpha cells); insulin flows past alpha cells where itinhibits glucagon secretion

b. Insulin/glucagon ratio (I/G) ratio is a determining factor in balancingmetabolism

c. Diabetics have poor glucose controlthat can be seen in fasting glucoselevels of glucose tolerance tests

i. Insulin/glucagon ratio is low in diabetesii. Low Insulinglucose is not absorbed

1. Delay in glucose absorption or decrease in plasma glucoseiii. Glucagon stimulates glycogenolysis (initially) AND gluconeogenesis

Adaptor

Lipid Kinase

2nd Messenger:PIP

Phosphatidylinosito

Protein kinases

Ras

Transcription

Protein kinases

Insulin Receptor is a tyrosine kinase

Lipid 2nd Messenger:PIP3-

Phosphatidylinositol

3,4,5trisphosphate

Protein kinases

Insulin Establishes Multiple Signal Cascades

How Does Insulin Signal??

Cell Membrane

Ras

A Protein Phosphatase is

one important target.

Adaptor

Alterations in

transcription

Alterations in

transcription


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iv. Result: Higher baseline for resting blood glucosed. Insulin acts to reduce blood glucose

i. Promotes:1. Glycogen synthesis2. Glucose uptake into

tissues3. FA biosynthesis andstorage as triacylglycerol

4. Glycolysis in the liver &acetyl CoA formation

5. LPL and lipid transfer toadipocytes

6. Amino acid uptake intocells increases proteinsynthesis

ii. Inhibits:1.

Glycogenolysis (glycogen phosphorylase)2. Gluconeogenesis

3. HSL (therefore, increasing lipid deposition)5. Insulin Affects Carbohydrate and Fat Metabolism

a. decreasesblood glucose by increasing uptake in muscle and adipose cellsb. decreasesglycogen phosphorylase and increases glycogen synthetase activity;c. decreasesgluconeogenic reactions;d. increasesglycolysis in the liver, increasing acetyl CoA formation;e. increasesfatty acid synthesis in the liverf. increaseslipoprotein lipase, increasing lipid transfer to adipocytes;g. increasesglucose in adipocytes, increasing triacylglycerol synthesis;h. decreaseshormone-sensitive lipase, increasing lipid depositioni. increasesamino acid uptake into cells, increasing protein synthesis

6. Insulin resistancea. Results from:

i. Genetics (e.g. IRS-1 mutation)ii. Changes in free fatty acid levels

iii. Proinflammatory cytokines by macrophagesiv. Alterations in adipokines (e.g adiponectin and resistin)

1. Resistin: confers insulin resistance2. Adiponectin increases sensitivity

v. Regulating Sensitivity to Insulin Signalingb. Loss of Insulin:

i. Increase glycogen breakdownto increase glucose1. Phosphorylate and stimulate glycogen phosphorylase for

breakdown2. Phosphorylate and inhibit glycogen synthase for synthesis

ii. Net increase in gluconeogenesisiii. Increase lipolysis(no phosphorylation of hormone sensitive lipase)

GLUCOSE

GLYCOGEN

GLUCOSE

FATTY ACIDS

TRIACYLGLYCEROLS

FATTY ACIDS

GLYCOGEN

LIVER

SKELETAL MUSCLE

ADIPOCYTES

Insulin Acts to Reduce Blood Glucose in 3 Major Ways

+

+

+

KETONE BODIES KETOSIS

AMINOACIDS

KIDNEY, dehydration

UPTAKE

VLDL

LACTATE

-GLYCOGEN

-GLUCONEO

Increasesglucose transport

(e.g fat and musc


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1. FAs give increased triglycerides, but ketone bodies are thecause of diabetic ketosis

2. Increases TG, increases a form of LDL, decreases HDLa. Hypertriacylglycerolemia

i. The lipid triad: elevated triglycerides, low HDL,small, dense low-density lipoprotein (LDL)c. Hyperglycemia:

i. Results from loss of insulinii. Without insulin, GLUT4 is not translocated to the plasma membrane

of fat and muscle cells and glucose uptake is impairediii. Glucose reacts with metals to generate reactive oxygen species (ROS)iv. The polyol pathway generates sorbitol and affects the

NADPH/NADP+ ratio1. NADPH is primarily used for biosynthesis; hyperglycemia

affects biosynthesis pathways ratesv. Advanced glycation end products(AGE) contributes to vascular

damage1. Elevated glucose gives protein glycation (stable attachment)2. Hb glycation gives carbohydrate history (Average level of CHO over lifespan of Hb)

a. Hemoglobin A1c integrated history: tells you well how well the patientis controlling the disease

b. Normal HbA1c = 4-6%c. Diabetic 7-8%

7. Complications of diabetesa. Diabetic RETINOPATHYb. Diabetic NEUROPATHY

i. Diabetic ketoacidosis (DKA): this coma is a medical emergency1. Intravenous fluids, insulin, and administration of potassium and sodium

ii. Hyperosmolar coma: plenty of intravenous fluids, insulin, potassium and sodium given assoon as possible

iii. Hypoglycaemic coma: administration of the hormone glucagon to reverse the effects ofinsulin, or glucose given intravenouslyc. Diabetic NEPHROPATHY

i. 30-50% develop kidney disease, proteinuriaii. Kidneys respond to high levels of blood glucose by doing their best to

excrete it, along with a great deal of waterd. Cardiovascular complications

i. Coronary artery disease risk 4 times greater, hypertensionFUEL METABOLISM DURING SEVERE UNTREATED

DIABETES

TISSUES BLOOD LIVER

ATP + CO2

STORED FUELS

Adipose Fat

Muscle Protein

KETONE BODIES

FATTY ACIDS

GLYCEROL

AMINO ACIDS

GLUCOSE

KETONE BODIES

Ac-CoA ATP + CO2

GLUCOSE

GLUCOSE LACTATE

ATP + CO2

LACTATE

URINE


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Exam IV Review

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