biomed sci new slides

8/11/2019 Biomed Sci New Slides

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Q.What is the function of Metabolism?

(a) To obtain and trap chemical energy from

substrates

(b) To build precursors to macromolecules from

substrates

(c) To assemble precursors into macromolecules.

Ex: DNA, Glycogen, Fat

(d) To degrade macromolecules into simpler

molecules


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Differences between Catab & Anab

Enzymes-allows for regulation and direction.Many enzymes may be same in a reversiblemetabolic pathway but some will differ

Energetics- ATP made in Catab; used for Anab

Cofactors-NAD NADHused for catabolism

NAD(P)HNADP occurs for anabolism

Cellular localization may differ e.g cyto vsmito


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REGULATION of Metabolism

1. Availability & concentration of substrates and

COFACTORS. Need to regenerate cofactors

2.Availability/Need for ATP

3. Enzyme characteristics-heme,metal, dimers

4.Regulatory enzymes-often allosteric. ATP

catabolic reactions while ADP them.

Product inhibiton of anabolic reactions

5.Genetic control of amount of enzyme in cell.Constitutive VS adaptive enzymes.

6. Hormonal regulation- chemical messenger

which or a metabolic reaction in another cell.


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ENZYME RELATIVE Keq MASS/ACTION

ACTIVITY RATIO

____________________________________________________________________

Hexokinase 1 4000 0.08

Phosphoglucoisomerase 117 0.36 0.24

Phosphofructokinase 17 1000 0.03

Aldolase 52 0.0001 0.00001

Triosephosphate isomerase 1768 0.40 0.24

Glyceraldehyde 3P DH 295 1000 900

Phosphoglyceromutase 67 0.10 0.12

Enolase 105 4.0 1.4

Pyruvate Kinase 258 1000 40


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CARBOHYDRATES/ GLUCOSE

Source of energy: Storage form of energy

Produces- fatty acids, cholesterol, steroids, someamino acids e.g serine, glycine, alanine

Produces pentoses for RNA and DNA and cofactors e.g

NAD, NADP, FAD, Vitamin B12.

Glucose- C6H12O6, a hexose as is fructose. Glucose is analdose, fructose is a ketose. Ribose is a pentose ,

glyceraldehyde is a triose. These are monosaccharides.Sucrose is a disaccharide of G and F. lactose is adisaccharide of G and galactose. Polysaccharides madeup of many G


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Glucose transporters and carriers

Passive carrier mediated glucose transport: No energy, G

moves down its conc. gradient. Blood glucose levels 4-8 mM.

GLUT 1 and 3 : Km= 1mM (high affinity), basal G uptake foralmost all tissues-RBC, brain, liver, heart etc

GLUT 2: Km= 15-20 mM (low affinity), senses high glucoselevels. Present in liver & pancreas

GLUT 4: present in adipose and muscle. Km = 5 mM. Amountis increased when INSULINtriggers translocation from Golgito plasma membrane-one major action for insulin

Active Transport move molecules against their conc gradient-require ATP or energy source/ gradient

GLUT 5: active transport linked to Na+ transport by the Gutand kidney


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SUMMARY SLIDE!

Tissue Affinity

Insulin Reg Pass/Active

GLUT1 So many! HIGH Nope. Passive

GLUT2 Liver, Pancreas low NO! Passive

GLUT3 So many! HIGH Nope. Passive

GLUT4 Muscle, Fat tbd YES!! Passive

GLUT5 GI tract

Kidney

Varies Yes Passive

SGLT1 GI tract

Kidney

HIGH Yes ACTIVE

SGLT2 GI tract

Kidney

low Yes ACTIVE


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INSULIN

Released by cells of the pancreas in response tohigh glucose. Function is to promote the use ofglucose by:

Increasing GLUT 4: glycolysis

( gluconeogenesis), glycogen synthesis (glyc bkdown), fatty acid synthesis and proteinsynthesis (their bkdown).

It works by activating or inhibiting rate-limitingenzymes in these pathways or increasing ordecreasing synthesis of these enzymes-posttranscriptional and transcriptional actions.


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GLUCAGON/ EPINEPHRINE

Glucagon is made by the cells of the pancreas.Epi is made by the adrenals (later lecture).Glucagon is released in response to low blood

glucose, Epi is response to stress. Their functionsare to increase production of glucose especiallyfor use by brain, RBC, muscle/heart.

They do this by gluconeogenesis and glycogen/

fat/ protein breakdown. Basically the opposite ofinsulin shown previously.

GLUCAGONworks in liver & adipose, Epi-all tissues


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GLYCOLYSIS

C6H12O6 + 2 NAD+ 2 ADP + 2 Pi2CH3COCOOH (pyruvate or pyruvic acid)+ 2ATP + 2 NADH. Irreversible, occurs in cytosol

of all cells. Pyruvate will be furthermetabolized

Only source of energy for RBC, major sourcefor embryonic tissue, retina, adrenals, someimmune cells , exercising muscle.

NOTE: need to regenerate NAD from NADH.

The 10 reactions of glycolysis


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The 10 reactions of glycolysis

Overall glycolysis reaction

Glucose + 2NAD++ 2ADP + 2Pi

2Pyruvate + 2NADH + 2H++ 2ATP + 2H2O

= irreversible steps in glycolysis*

98

7

6

54

21

*

3*

*10


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Hexokinase VS Glucokinase

Normal blood G conc 4-8 mM. 10-15mM aftermeals

Glucose + ATP G6P + ADP Irreversible

HK - low Km for G ( 0.1-1 mM), in all cells, byproducts G6P, ADP, reacts with other hexoses,not affected by insulin, glucagon, Epi

GK- high Km for G ( 10 mM), in liver & pancreas,not affected by products, specific for G, mRNAlevels by insulin,byglucagon & Epi


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PHOPHOFRUCTO-1-KINASE (PF-1K)

F6P + ATP F1,6 Bis P + ADP Irreversible

(+) allosteric effectors-AMP, Pi, NH4, F1,6 bis P

(-) allosteric effectors- ATP, citrate

MAJOR (+) effector is F2,6 Bis P

F6P + ATP F2,6 Bis P + ADP F2,6 Bis P kinase RX

F2,6 Bis P F6P + Pi F2,6 Bis P phophatase RX

Insulin kinase butPhase. This will F2,6 andthen PF-1K. Glucagon/ Epi kinase butPhase,this willF2,6 and then PF-1K. Covalent modif

St 3


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Step 3:

-F6P converted to fructose 1,6-bisphosphate

(F16bP)

-Enzyme: Phosphofructokinase

-RATE LIMITING STEP

-Requires ATP so irreversible

-2 substrate binding sites

-Inhibitors: ATP, citrate

-Allosteric

-Activators: AMP, Pi, NH4+, F16bP, (F26bP)

-Allosteric

-Also hormonal regulation-F6P goes into side rxn to make F26bP

which is a major regulator of PFK

-Insulin leads to dephosphorylation of

F26kinase and F26phosphatase which

means more F26bP is produced and PFK is

upregulated-Insulin indicates fed state, it tells

the body there is lots of glucose so it

stimulates glycolysis to use this up

-Glucagon/epi, via cAMP-PKA,

promote phosphorylated inactive

state of kinase and phosphorylatedactive state of phosphatase

1,3-bisphosphoglycerate


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ALDOLASE

F1,6 Bis P G3P + DHAP

DHAP can alpha glycero P, very important fortriglyceride and phospholipid synthesis or G3P

( triose P isomerase RX) and continue glycolysis.Hence, F1,6 Bis P 2 G3P and we need to X by 2all the next steps of glycolysis.

NOTE: when F1,6 Bis P 2 G3P, C1 of the

original G is equivalent to C6, C2and C5 cannot bedistinguished and C3 and C4 cannot bediscriminated from each other. Try to prove this.


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G3P DH REACTION

G3P + NAD +Pi 1,3 Bis P glyceric acid + NADH

1,3 Bis PGA is a high energy compound. The nextstep ( PGK ) ATP: G3P + ADP 3PGA + ATP. Thisis substrate level phosphorylationno need formito, ets, oxygen. Arsenic uncouples

G3PDH by heavy metals-Hg, Cd,PB. We oxidizedan aldehyde (G3P) to an acid (3PGA) in these 2coupled steps. The energy was trapped as a high

energy intermediate 1,3 Bis PGA, not released asheat if G3P 3PGA directly.

1,3 Bis PGA can be mutated to 2,3 Bis PGA (HB-O2)


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Pyruvate Kinase

PEP + ADP Pyr + ATP. Irreversible. PEP is a highenergy compound. Substrate level phosphoryl.Requires monovalent cations like K or Na.

PK ATP, NADH, Acetyl CoA and Glucagon- or epi-mediated Phosphorylation. by F1,6 Bis P andinsulin stimulated dephosph.

NOTE: ATP was made here & the G3P/ 3PGA

coupled steps. We made 4 ATPs ( 2 per triose)and used 2 ATPs for the HK and PFK steps, net is 2ATP/Glucose.


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FATES OF PYRUVATE

RBC, exercising muscle, embryonic tissuelactate via lactate dehydrogenase (LDH)

Yeast , conversion to ethanolvia PDC & ADH

Aerobic cells, conversion to acetyl CoA via PDH:Pyr + NAD+ CoA Acetyl CoA + CO2+ NADH

FATES of Acetyl CoA CO2 via TCA Cycle, ATP;

citrate which fatty acids ketones bodies,cholesterol, steroids

Acetylation RX e.g histones


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Lactate Dehydrogenase

Rxn: pyruvate+NADH+H+Lactate + NAD+ Enzyme: lactate dehydrogenase (LDH)

Reversible

Anaerobic RBC have no mitochondria so this is their only

way to get NADH reoxidized back to NAD+(req.for glycolysis, their only source of ATP)

Net rxn: glucose +2ADP+Pi2 lactate +2ATP LDH Tetramer: 2H & 2M chainso 5 isoforms:H4, H3M, H2M2, HM3 and M4

H4 and H3M: Brain or Heart M4 and M3H: RBC & skeletal muscle

H subunits have high affinity for lactate &NAD+

;pyruvate --| H4


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dehydrogenase


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TCA CYCLE

In mito of all aerobic cells. IRREVERSIBLE

Acetyl CoA + 3 NAD + FAD+ GDP+ Pi+ 2 H2O

2 CO2 +3 NADH+FADH2+GTP+CoASHENERGETICS: 3 NADH 7.5 ATP; GTP=ATP

FADH2 1.5 ATP 10 ATP/Acetyl CoA

Of the 6 carbons in G, C3,4 CO2 in PDH RXC1,2,5,6CO2 in TCA cycle


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STEP 41.Kg + NAD++ CoA-SH Succinyl-CoA+ NADH + H+

+CO21.Kg Dehydrogenase is a complex of enzymes

2.E1= Kg Decarboxylase has Thiamine PP as cofactor1.inhibited by ATP

3.E2 = uses lipoic acid1.inhibited by succinyl CoA

4.E3 = uses FAD & NAD to regenerate Lipoic acid1.inhibited by NADH


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Step 5

1.Succinyl CoA + GDP +PiSuccinate +CoA-SH +GTP1.Enzyme = Succinyl CoA synthase (Thiokinase)2.CEDERBAUM WORKED ON THIS enzyme

(and he is adorable)

St 6 7 d 8


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Steps 6, 7 , and 81.Succinate + FAD Fumarate + FADH2

1.Enzyme: Succinate dehydrogenase

2.Fumarate + H20 Malate1.Enzyme: Fumarase

3.Malate +NAD+OAA + NADH + H+

1.Enzyme: Malate dehydrogenase

*each rxn is reversible butMalate dehydrogenase favorsOAAMALATE


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ANAPLEROTIC (NOT EROTIC) RX

Intermediates of the TCA cycle can be pulled outinto other RX and pathways. OAA will not beregenerated , TCA stops.

Anaplerotic RX are replenishing RX, fill in thepulled out intermediates.

#1 in next slide is pyr carboxylase (PC), only in mito

Not shown is the PEP carboxykinase RX: PEP +CO2+ GDP OAA + GTP (PEPCK in mito and/orcyto)

PC & PEPCK very important for gluconeogenesis


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Final Products

2 CO2s 1 FADH2(= 1.5 ATP) 3 NADH + 3H+(= 7.5 ATP) 1 GTP (= 1ATP)

Total = 10 ATP/ Acetyl CoA


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OVERALL YIELD!


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Mitochondrial Carriers

Carrier Description

Phosphate exchanges Pi with OH

Dicarboxylate exchanges Pi/malate/succinate for

eachother

Tricarboxylate exchanges citrate, isocitrate, malateor PEP for eachother

Alpha Ketoglutarate exchanges alphaKG for malate

Pyruvate exchanges pyruvate for OH or ketone

bodies

Glutamate exchanges glutamate for OH

Aspartate exchanges asparate for glutamate

Adenine nucleotide exchanges ADP for ATP


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SHUTTLES

NADH and NADPH cannot enter or leave mito

Need substrate shuttles to transport them. Mostimp. for NADH are the -glycerophosphate (GP)

and malate-aspartate (MA) shuttles. These arealso used for metabolism of other RX producingNAD(P)H e.g ethanol.

Transamination is the transfer of an alpha amino of

one amino acid to a keto acceptor to new aaand a new keto acceptor

How to distinguish between the GP & MA shuttles


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Cytosol Mitochondrial matrix

Kg+CO2

carrier 4

cyto

ICDH mito

ICDH

carrier 3

isocitrateisocitrate

NADP+

NADPH

NADP+

NADPH NAD+

NADH

transhydro-genase

Kg+CO2


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GLYCOGEN METABOLISM

Glycogen a storage form of G, made up of many Gunits linked by 1C of one G to the C4 of anotherG. Also C1 to C6 links make branches.

Glycogen made underhigh- energy, carbs & GGlycogen broken downlow- energy, carbs & G :

vigorous exercise/ stress: diabetes

Stored mainly in liver and muscle. Function of liver

Glyc is to produce G for use by other tissuesneeding energy. Function of muscle Glyc is toproduce G for use of muscle itself


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Glycogen Breakdown

Catalyzed by phosphorylase and debranchingenzyme many G1P + 1 G where a branchpoint

G1PG6P via phosphoglucomutase

G6PG via G6Pase in liver OR glycolysis inmuscle. Note that G6Pase is by glucagon/Epi

Phosphorylase is activated when P byphosphorylase kinase (PK) which itself isactivated by cAMP-dependent PKA. Thus

glucagon in liver and Epi in liver and muscleGlyc breakdown. Insulin promotes the dePstate of PK and phosphorylase which activities


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Glycogen Synthesis

GG6PG1PUDPG. Need exisiting primer-

Glycogen (n) + UDPG Glycogen ( n+1) + UDP

Catalyzed by Glyc Synthase ( GS) and Branching enzyme

GS active in de P state (GS-OH) and inactive when P(GS-OP). Insulin, via protein phosphatase 1 (PP1)promotes the de P state while cAMP PKA promotesthe P state. Same with PK and phosphorylase. NotedeP state GS and Glyc synthesis but PK and

Glyc breakdown-insulin while P state PK andPhosphoryalse & Glyc breakdown, but GS Glucaon/Epi


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MORE GLYCOGEN REGULATION

I am kind of tired. Need coffee,a PDE inhibitorAMP Phos-OH ( low activity) which will activity a bit

and some Glyc breakdown.

ATP & G6PPhos-OH

G6P GS-OP ( low activity) which will some Glycsynthesis

Glucaon/Epi PP1 by P and also by activity of a PP1inhibitor by P. InsulinPP1 by deP and by also byinactivating the PP1 inhibitor.

Liver Phos is a glucose sensor-binds PP1 when G is notpresent so PP1 not effective but releases PP1 when Gis present so now PP1 starts to do its job


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PENTOSE P PATHWAY

Function s to produce NADPH, ribose 5 P,interconversions of C3,C4, C5, C6 and C7 sugars.

Active in cytosol of liver, RBC, adipose, adrenals, mammary tissue

Regulated by Conc. of G6P, NADP/NADPH. Insulin small G6PDH

First 2 steps catalyzed by G6PDH & 6PGADH- IRREV

3 G6P3CO2+6 NADPH +3Ribulose 5P. Step 3 is

3 Ribulose 5P3 Ribose 5P PPIsomerase-REVERSIBLE.

Rearrangements: Transketolase,Transaldolase,Transketolase RX

3 Ribose 5P2F6P+ G3P(=0.5 G6P); 2.5 G6P made. TPP cofactor for TK

NOTE G6P & G3P can R5P by reversing Rearrangement RX

CO2 came off C1 of G6P, can come off other Cs after rearrangement


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OXIDATIVE STRESS & PPP

Hydrogen peroxide, H2O2,made in aerobic cellsfrom mito ets or autooxidation of ferroushemes like hemoglobin. H2O2 removed by

catalase and by glutathione peroxidase, GPX,which uses glutathione,GSH, a tripeptide.

H2O2 + 2 GSH 2 H2O + GSSG GPX RX

Note GSH is oxidized to GSSG and must bereduced back to GSH by glutathione reductase

GSSG + 2NADPH2 GSH +2 NADP


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G6PDH DEFICIENCY

Most common inborn error of metabolism

Complete deficiency is embryonic lethal.

Certain drugs G6PDH, can RBC hemolysis,

anemia. Those with low G6PDH sensitive tooxidant stress produced by drugs, chemicals,foods (Fava beans), high O2, high altitudes

Wernicke Korsakoff Syndrome- poor memory,orientation,gait, mental function/ neuropsyc.Disorders. Due to 10X in Km for TPP by TK


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GLUCONEOGENESIS

Occurs with low G/CH diet,fasting/starvation,whenG is needed for quick & rapid energy for muscle.

Liver & kidney. Compartmentalized cyto & mito.

Most common substrates-lactate & aapyr.

2 Pyr+ 2 GTP +4 ATP+ 2 NADHG + 2 GDP+ 4 ADP +2 NAD + 6 Pi. Where is energy from?

Most, but not all, steps are reversal of glycolysis.

To bypass PK, PF-1K, & GK/HK, need gluconeoenzymes PC, PEPCK, F1,6 Bis Phase, G6Pase These

are by glucagon/Epi and by insulin.


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Step 2&3: Pyruvate OAA PEP

Pyruvate converted to PEP viatwo steps to bypass theirreversible pyruvate kinasereaction

Step 2: Pyruvate carboxylase

reaction Pyruvate + ATP + CO2 OAA

+ ADP +Pi

Happens in mito

Step 3: PEP carboxykinase

(PEPCK) reaction OAA + GTP PEP + CO2 +

GDP

Happens in mito and cytosol


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Step 6&7: F6P G6P glucose

- F1,6 bisphosphatase:

stimulated by low

energy signals

(AMP, Pi, NH4+,

glucagon, epi) andinhibited by high

energy signals (ATP,

citrate, insulin)

- G6Pase: stimulated

by glucagon andepi, inhibited by

insulin


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CORI CYCLE

or exercising muscle


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REGULATION


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FATTY ACIDS

Functions:

Major source of energy for most tissues but notRBC or brain

Major storage from of energy as triglyceridesMake up phospholipids & glycolipids of biological

membranes

Produce signal transduction molecules e.g inositol

Phosphates, Diacylglycerol

Produce prostaglandins, leukotrienes


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FA OXIDATION

Palmitoyl CoA: C16+7 FAD+7 NAD+7CoA+7H2O

8 Acetyl CoA +7 FADH2 + 7 NADH:

Energetics: 8 Acetyl CoA 80 ATP7 FADH2 10.5 ATP 7 NADH17.5 ATP

Total 108 ATP made2 ATP to activate =106ATP

Occurs in Mito & some in peroxisomes


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id i h


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-oxidation pathway

4 enzyme catalyzedreactions to remove 2carbon fragment at atimeproduce acetylCoA plus a new fattyacyl CoA shortened by2 carbon atoms

Acetyl CoA can thenenter Kreb cycle

Repeats until all thecarbons are oxidized toacetyl CoA

Each round produces:one FADH2 and oneNADH Ex. C18 fatty acid will

undergo 8 rounds

ODD CHAIN FA


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ODD CHAIN FA

Basically same as even chain FA- -oxidation of

C17 FA CoA 7 Acetyl CoA +Propionyl CoA

CH3CH2COCoA+ CO2D-MethylmalonylCoA via

propionyl CoA Carboxylase ( Biotin) L-MMCoA

via MMA racemase Succinyl CoA via MMA

Mutase. Mutase requires deoxyadenosyl B12

to catalyze this remarkable rearrangement RX

Will return to this in our B12 lecture


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R l ti f id ti


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Regulation of -oxidation

Availability of fatty acids via activatedhormone sensitive lipase

Availability of carnitine.

Malonyl CoA, the product of the rate-limiting

enzyme of fatty acid synthesis (next lecture)

inhibits carnitine acyl transferases 1 and 2.

Rate of the electron transport chain

Q: Explain possible major problems for patients A B C in oxidizing palmitate


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Q: Explain possible major problems for patients A, B, C in oxidizing palmitate

into CO2:

Substrate Rate of CO2 Production

Control A B C

Palmitate 100% 10% 10% 10%

Palmitoyl-coA 100% 100% 10% 10%

Palmitoyl-Carnitine 100% 100% 100% 10%

Acetyl-coA 100% 100% 100% 100%

FA SYNTHESIS


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FA SYNTHESIS

8 Acetyl CoA+ 7ATP+ 14NADPH

C16 palmitoyl CoA+7CoA +7ADP+7Pi + 14NADP

Compartmentalized between Mito & Cyto

Acetyl CoA from Pyr ( G) & AA ( not from FA)

NADPH from PPP, Transhydro & isocit shuttle &

malic enzyme

S b t t d M t b li C diti


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Substrates and Metabolic Conditions

Activating conditions High glucose

High protein

Insulin Activates SREBP

Inhibiting conditions

Glucagon Inhibits SREBP

Malonyl CoA Inhibits acyl carnitine transferase that brings FA into mito for B-oxidation

Location Cytoplasm of most cellsespecially liver

Substrate Acetyl CoA (transported to cytoplasm as citrate from glycolysis)

Energy demands ATP and NADPH


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Rate Limiting Step


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Rate Limiting Step

Acetyl CoA + ATP + CO2malonyl CoA + ADP + Pi Enzyme: Acetyl CoA carboxylase

Cofactor: biotin

Activators: Citrate

Insulin (dephos ACC and stimulating it)

Inhibitors:

palmitoyl CoA (product)

AMP activated kinase (phosphorylates ACC)

Glucagon/cAMP-PKA (phosphorylates ACC at another site)


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REGULATION


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REGULATION

Many of the lipogenic enzymesACC, FAS, ATP-citrate lyase, malic enzyme are induced by insulinwhich activates processing of the mastertranscriptional activator Sterol Regulatory

Element Binding Protein (SREBP)to its activeform.

High glucose activates Carbohydrate ResponseElement Binding Protein which upregulates manygenes involved in carbo and FA synthesis ( JCpaper).

FATTY ACID OXIDATION


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FATTY ACID OXIDATION

Three patients(ABC)have trouble ox Palm to CO2

CONTROL A B C

SUBSTRATE RATE OF CO2 PRODUCTION

Palmitate 100 10 10 10

Palm CoA 100 100 10 10

Palm Carnitine 100 100 100 10Acetyl CoA 100 100 100 100


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Fed State


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Basal State


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Starved State


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