Biochem Ii

Front 1

How is Fructose-2,6-bisphosphate regulated?

Back 1

By a single protein with 2 domains.

PFK II domain phosphorylates F6P -> F26BP

FBPaseII domain hydrolyzes F26BP -> F6P

This enzyme is regulated by a serine phosphorylation.

when glucose is low: PKA phosphorylates it to FBPase II (gluconeogenesis and stop glycolysis)

When there is excess glucose, PKA decreases and F6P increases. F6P activates phosphoprotein phosphatase which dephosphorylates the enzyme and the enzyme becomes PFK II (glycolysis on and gluconeogenesis off)

Front 2

Glucagon's effects

Back 2

released when glucose is scarce by alpha pancreatic cells. Activates adenylyl cyclase-->cAMP--> stimulates PKA to deactivate pyruvate kinase and favor gluconeogenesis.

Front 3

substrate cycles:

what are they and why are they important?

(I.E. not "futile")

Back 3

A pair of reactions that go the opposite direction between 2 substrates.

They are important AND NOT FUTILE for 2 reasons:

- they allow the amplification of metabolic signals (can change the overall net flux with smaller individual changes)

- they also help generate heat

- also makes sure there are no dead spots where there is no control (turn off one and the other on)

Front 4

Cori Cycle

Back 4

Lactate formed in active muscle is released into the blood and can be converted back to glucose by the liver and released into the blood.

this cycle shifts part of the metabolic burden of active muscle to the liver.

Front 5

What are the 2 sources of energy in the cell?

Back 5

-Glycolysis
-Oxidative Phosphorylation

Front 6

How is Acetyl CoA synthesized?

Back 6

Either from pyruvate (with P.D.H) or from Fatty Acid Oxidation

Front 7

How is NTP, DNA, or RNA synthesized?

Back 7

Through the Pentose-Phosphate pathway

Front 8

How can one change the rate of a reaction?

Back 8

- change concentration of Enzyme
If enzyme level decreases, then rate decreases

- change concentration of Substrate
as long as substrate does not saturate enzymes, than the rate of reaction will change.

- activate/inhibit enzymes (reversibly)

- activate/inhibit enzymes (irreversibly)

- protein-protein interactions

Front 9

Differences between hexokinase and glucokinase?

Back 9

Location:
-glucokinase is found only in the liver
-hexokinase is found everywhere

Specificity:
hexokinase: phosphorylates most carbs
glucokinase: works only on glucose

Adaptive:
hexokinase: no since it has such a low Km
glucokinase: yes with a high Km (important for function)

Inhibition by ADP and G6P?
hexokianse: yes
glucokinase: no

Front 10

Why are there 2 different pathways between the synthesis and degradation of a molecule?

Back 10

Because if they were one reaction, there would be no control.

Front 11

Why is ATP high energy?

Back 11

- it has 4 negative charges which dont like to stay together.
- resonance stabilization of the products
- pKa is ~ 7 so need neutral pH or else the high energy is gone

Front 12

Why is PEP such a high energy molecule?

Back 12

If you were to remove the phosphate, the keto-tautomer is very stable and that drives the reaction.

Front 13

For (CH2O)n why does n have to be at least 3?

Back 13

Because there needs to be at least 2 hydroxyl groups and 1 carboxyl (ketone or aldehyde)

Front 14

stereo isomers

Back 14

Isomers that differ only in the way atoms are oriented but the same structure (mammals always have D carbs and L proteins)

Front 15

"D" stereo isomers

Back 15

carbs that have the OH group on the last C (from the carboxyl group) facing to the right

Front 16

why is high glucose bad?

Back 16

because glucose is very reactive in linear form and can cause unwanted reactions (nonenzymatic protein glycosylations)

- can test by measuring hemoglobin A-1-C which is glycosylated with glucose

Front 17

glycolysis cycle

Back 17

glucose --> hexo/glucokinase --> Glucose-6-Phopshate --> phosphoglucoisomerase --> Fructose-6-Phosphate --> phosphofructokinase --> Fructose 1,6 BP --> aldolase --> dihydroxyacetone phosphate + glyceraldehyde-3-P (DHAP -> triose-phosphate isomerase -> G3P) --> G3P dehydrogenase --> 1,3 bisphosphoglycerate --> phosphoglycerate kinase --> 3P Glycerate --> phosphoglycerate mutase --> 2P Glycerate --> enolase --> PEP --> pyruvate kinase --> Pyruvate

Front 18

Principle fate of glucose in the cell

Back 18

phosphorylated by hexokinase into G6P (glucose 6 phosphate). This traps the glucose in the cell and makes it more reactive

Front 19

overall substrate/reactant reaction in glycolysis

Back 19

1 Glucose + 2 ATP + 2 Pi + 2 NAD + 4 ADP --> 2 Pyruvate + 4 ATP + 2 ADP + 2 NADH + H2O

Front 20

Main regulating enzymes of glycolysis

Back 20

Hexokinase

Phosphofructokinase (PFK)

Pyruvate Kinase

Front 21

What is necessary for proper kinase function?

Back 21

divalent metal cation (such as Mg) to form a complex with the ATP

Front 22

Mechanism of hexokinase function

Back 22

when glucose binds, hk changes conformation to surround glucose (induced fit). Forms non-polar environment around glucose to favor Pi donation and prevents water from entering and breaking up ATP without Pi donation.

Front 23

Why does glucose need to get isomerized to F-1,6-BP before the aldol cleavage?

Back 23

Because otherwise the aldol cleavage would have made 2C and 4C products and two pathways would be needed to extract energy.

Front 24

Mechanism of TPI (Triose phosphate Isomerase)

Back 24

isomerizes DHAP to G3P through an enediol intermediate and prevents side reactions from occuring by keeping active site closed until the right reaction takes place.

Front 25

Mechanism of G3P D.H. (glyceraldehyde-3-P-Dehydrogenase)

Back 25

redox rxn is coupled to Pi substitution by thioester intermediate. Redox is favorable and Pi addition is unfavorable so the redox reaction has thioester intermediate which facilitates the Pi attack and decreasing activation energy.

Front 26

substrate level phosphorylation

Back 26

a reaction where a phosphate group is transferred from a substrate with high phosphoryl-transfer potential to a lower one. (As opposed to ATP formation from ionic gradients)

Front 27

3 Fates of Pyruvate

Back 27

1) made into ethanol

- pyruvate --> pyruvate decarboxylase --> acetaldehyde --> alcohol dehydrogenase --> ethanol

2) Lactate

- pyruvate --> lactate dehydrogenase --> lactate

3) Acetyl CoA --> Oxidation

Front 28

how does galactose enter glycolysis?

Back 28

galactose --> galactokinase --> galactose-1-P + UDP-glucose --> galactose-1-P-uridyl transferase --> UDP galactose + glucose-1-P.

glucose-1-P is isomerized to G6P through phosphoglucomutase.

UDP Galactose is converted back to UDP glucose through UDP-galactose-4-epimerase to work another galactose.

Front 29

How can the cell produce more galactose if needed?

Back 29

through the reverse reaction of UDP-glucose --> UDP galactose (UDP-galactose-4-epimerase)

Front 30

PFK regulation

Back 30

the most important regulated enzyme in metabolism.

regulated by energy state of the cell... the ATP/AMP ratio. ATP allosterically inhibits.

Also regulated by pH in muscle cells. When lactate levels elevate, pH decreases and signals glycolysis to cease. pH regulation is absent from liver since lactate is not formed in significant amounts.

Also activated allosterically by F2,6-BP.

Inhibited by citrate in the liver. citrate enhances ATP's inhibition.

Front 31

Why is AMP the signal for a low energy state of the cell (as opposed to ADP)?

Back 31

1) ADP can be further metabolized into ATP and AMP

2) AMP levels are the lowest in the cell ([ATP]>>[ADP]>>[AMP]). so small changes in ATP lead to larger changes in AMP. Thus AMP provides a greater sensitivity to cellular energy levels.

Front 32

Hexokinase regulation and how it differs from glucokinase regulation

Back 32

G6P inhibits enzymatic activity

(NOT an inhibitor of glucokinase since glucokinase's affinity for glucose is 50X lower so that brain and muscles get first call when supply is limited)

Front 33

Pyruvate Kinase regulation

Back 33

regulated by energy state of cell (ATP/AMP)

Also activated directly by F1,6-BP. [dependence]

Inhibited directly by Alanine

L isozyme (liver) can be phosphorylated by PKA and inactivated

M isozyme (muscle) cannot be reversibly phosphrylated

Front 34

Glucose Transporters and their properties:

Back 34

GLUT 1 and 3: basal glucose uptake in all cells (very low Km)

GLUT 2: liver and B pancreatic cells. very high Km so glucose only uses these transporters when there is much glucose in blood. helps liver and pancreas sense glucose levels of blood and react w/ insulin and glycogen synthesis

GLUT 4: medium Km. activated by insulin. found in muscle and fat cells. exercise can increase amount of these GLUTs

GLUT 5: small intestine. insulin independent. fructose transporter

Front 35

what precursors can be used for gluconeogenesis?

Back 35

lactate, amino acids, glycerol

lactate --> pyruvate --> glucose

glycerol --> glycerol kinase --> glycerol phosphate --> glycerol phosphate D.H. --> DHAP --> glucose

amino acid --> OAA --> PEP --> glucose

Front 36

Where are enzymes for gluconeogenesis found?

Back 36

most are in cytoplasm.

pyruvate carboxylase: mitochondria

glucose-6-phosphatase: ER

Front 37

gluconeogenesis pathway

Back 37

pyruvate --> pyruvate carboxylase --> OAA --> PEP carboxykinase --> PEP --> 2PG --> 3PG --> 1,3BPG --> G3P --> DHAP --> F1,6BP --> F1,6 Bisphosphatase --> F6P --> G6P --> G-6-phosphatase --> glucose

Most is kept as G6P so glucose cannot leave the cell.

G6P phosphatase is only present in liver and kidneys and is tightly regulated

Front 38

where does lactate, amino acids, and glycerol enter gluconeogenesis?

Back 38

lactate --> lactate D.H. --> pyruvate


glycerol --> glycerol kinase --> glycerol-P --> glycerol-P-D.H. --> DHAP

Front 39

is gluconeogenesis the reversal of glycolysis

Back 39

NO because...

-PFK, HK, and Pyruvate Kinase are irreversable enzymes

-the (G) of glu-pyr = -84kJ

- pyruvate carboxylase, PEP carboxykinase and the 2 phosphatases work around the forward enzymes

Front 40

Mechanism of Pyruvate Carboxylase

Back 40

Uses Biotin and CO2.

the biotin serves as a carrier of activated CO2 and is attached to enzyme in a long flexible chain.

biotin allows the CO2 to be transferred without input of energy

formation of carboxy-biotin is dependent on the presence of Acetyl CoA.

this makes sense because gluconeogenesis requires alot of energy and you need to make sure that there is enough energy in the cell. excess Acetyl CoA means alot of energy in the cell (from b-oxidation)

Front 41

Mechanism of PEP carboxykinase

Back 41

OAA needs to be carboxylated so that the removal of carboxy group can drive the PEP formation.

ATP helps place CO2 on.

Front 42

How is G6P made into glucose

Back 42

can only be done in the liver or kidneys since G6P phosphatase is only present there.

G6P is transported to ER -> hydrolyzed to glucose -> transported back to cytoplasm

Front 43

stoichiometry of gluconeogenesis

Back 43

2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 6H2O --> glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD

(G) = -38kJ

Front 44

Draw glycolysis/gluconeogenesis regulation chart

Back 44

see p465

Front 45

Regulation of gluconeogenesis enzymes

Back 45

pyruvate carboxylase: inhibited by ADP; activated by Acetyl CoA

PEP carboxykinase: inhibited by ADP

F1,6BPhosphatase: inhibited by F-2,6-BP, AMP; activated by citrate

Front 46

Efffects of insulin on metabolism

Back 46

GLUT 4 go to plasma membrane and bring in more glucose for glycolysis/glycogen synthesis.

also stimulates expression of PFK, pyruvate kinase.

inhibits GSK to stimulate glycogen synthesis

Front 47

adenylate kinase

Back 47

catalyzes: ADP + ADP --> AMP + ATP

Front 48

iodoacetate

Back 48

classical inhibitor of glycolysis.

inhibits G3P D.H.

Front 49

main function of citric acid cycle

Back 49

harvest high-energy electrons from carbon fuels

Front 50

NAD

Back 50

nicotinamide adenine dinucleotide.

a known electron carrier that can accept and donate electrons

Front 51

pyruvate dehydrogenase complex

- reaction
- components
- mechanism

Back 51

an IRREVERSIBLE enzymatic complex that converts pyruvate to acetyl CoA.

Occurs in the mitochondria

analogous to α-keto-gluterate D.H.

3 enzymes and 5 coenzymes

- pyruvate D.H. (E1); dihydrolipoyl transacetylase (E2); dihydrolipoyl D.H. (E3)

- TPP; lipoamide; FAD; CoA; NAD

1) pyruvate + TPP -> E1 -> hydroxyethyl-TPP + CO2 [decarboxylation]

2) HE-TPP + lipoamide -> E1 -> TPP + acetyl-lipoamide [oxidation]

3) acetyl-lipoamide + CoA -> E2 -> Acetyl CoA + dihydrolipoamide [transfer to CoA]

4) Dihydrolipoamide + FAD -> E3 -> lipoamide + FADH2

4a) FADH2 + NAD -> E3 -> FAD + NADH
[regeneration of lipoamide and FAD]

The lipoamide arm swings to each E complex and the rxn.

Front 52

why is FAD unusual in Pyruvate D.H. complex?

Back 52

Because it usually has a lower electron-transfer potential than NAD so it would not want to donate. But FAD's association with the enzyme increases the electron transfer potential.

Front 53

synthase

Back 53

an enzyme that joins two units without the input of energy from a triphosphate

Front 54

why is proximity between enzymes in a complex important?

Back 54

increases the overall rate of reaction

minimizes side reactions

Front 55

citrate synthase:

-mechanism

Back 55

1) OAA binds and causes Acetyl CoA binding site to open and OAA gets trapped [induced fit]

Front 56

Vitamins and their corresponding co-enzymes

Back 56

B1 (Thiamine) -> TPP
B2 (Riboflavin) --> FAD
Niacin -> NAD
Pantothenate -> CoA

Front 57

if you were to radiolabel CO2 and study gluconeogenesis, would radiolabeled C appear in glucose?

Back 57

Yes because for OAA to leave the mitochondria it must first turn into malate. however, once it is malate, it can become fumerate and fumerate it loses its assymtry. now once it becomes OAA again, the top can be the bottom and the bottom can be the top so roughly half of the radiolabeled C will remain in the skeleton by chance

Front 58

overall reaction of oxidation and phosphorylation

Back 58

oxidation: NADH + O2 -> NAD + H2O
phosphorylation: ADP + Pi -> ATP

Front 59

what is respiratory control ratio

Back 59

rate (b) / rate (a) in oxidative phosphrylation

this is the dependence of respiration on ADP.

graph of O2 vs time. this is the

-slope of the graph without ADP (A)

-slope of the graph with ADP (B)

the ratio should be ~ 5.

in worst case scenario it is 1 (ADP has no effect)

Front 60

P/O ratio of NADH and FADH2

Back 60

ratio of ATP produced to O consumed.

NADH is about 3 and FADH2 is about 2.

there must be 3 sites and FADH2 only uses 2 of the sites

Front 61

in regular conditions, can you have oxidation without phosphorylation?

Back 61

no they are coupled. without one, the other cannot take place.

Front 62

2,4 dinitrophenol

Back 62

oxidative phosphorylation uncoupler.

if this is given to a mitochondria, O2 will be consumed but no ATP will be made since no ADP is present.

not an inhibitor because it is not inhibiting anything; rather it is stimulating O2 consumption. it is only freeing oxidation from phosphorylation

its mechanism of uncoupling is by helping protons leak across and destroying the gradient.

Front 63

what happens if you give:
1) ETC inhibitor
2) DNP

what happens if you give
1) phosphrylation inhibitor + ADP
2) DNP

Back 63

A) no change in O2 consumption because once the ETC is blocked, DNP cannot do anything.

B) first no change in O2 consumption because phosphorylation is inhibited, but then all O2 consumed since DNP uncouples and ETC can now function on its own.

Front 64

carriers of ETC

Back 64

1) Flavins (FMN)
2) nonheme irons (Fe-S) [cannot pick up protons]
3) heme proteins (cytochromes)
-these are colored and abundant in dark meat
[cannot pick up protons]
4) Co Q (ubiquinone)

Front 65

why is dark meat dark and light meat light?

Back 65

because dark meat is muscle tissues that were heavily used and thus had more mitochondria and more color

Front 66

order of ETC

Back 66

NADH reduces FP1 (FMN and Fe-S) [Fp1 is also called NADH-Q Reductase]

FP1 passes to CoQ

CoQ passes to cyt B to cyt C1 [QH2-cyt c reductase]

C1 passes to cyt C to cyt A [cyt c oxidase] to O2

Front 67

3 enzymes of ETC

Back 67

NADH-Q Reductase - FMN/Fe-S (FP1)

QH2 Cyt C Reductase - cyt B and cyt C

Cyt C oxidase - cyt a+a3

Front 68

FP1

FP2

FP3

FP4

Back 68

1) NADH Q reductase

2) succinate D.H. (same enzyme as in citric acid cycle)

3) glycerol-3-P D.H.

4) acyl-CoA D.H.

Front 69

ETC:

site 1 inhibitors
site 2 inhibitors
site 3 inhibitors

Back 69

1) rotenone, amytal [doesnt inhibit the whole ETC since CoQ is not inhibited!]
2) antimycin A
3) cyanide, CO, H2S, N3 (azide)

Front 70

ETC:

complex I, II, III, IV

Back 70

I: FP1 (NADH Q reductase)
II: FP2 (succinate D.H.)
III: QH2 cyt C redutase
IV: site C oxidase

Front 71

How to get NADH from cytosol to mitochondria

Back 71

1) glycerol-P-shuttle

2) malate-aspartate shuttle

Front 72

glycerol-P-shuttle

Back 72

way to get reducing equivalents to the mitochondria.

NADH + DHAP -> glycerol-P-D.H. (NAD-linked) -> NAD + glycerol-3-P

glycerol-3-P can enter the intermembrane space of the mitochondria and react with an FAD-linked glycerol-P-D.H. that is embedded in the inner membrane to reduce FAD to FADH2.

glycerol-3-P doesnt have to get shuttled into the matrix

The FAD-linked G-3-P D.H. is FP3 of the ETC.

also NOTE that you get 2/1.5 ATP from this shuttle per NADH instead of 3/2.5

Front 73

malate aspartate shuttle

Back 73

way to get reducing equivalents to the mitochondria

NADH + OAA -> MDH -> malate + NAD.

malate can freely enter the mitochondria through an antiporter that sends a-keto-glutarate out.

malate then enters the TCA cycle and makes OAA. OAA cannot exit the mitochondria so must be transaminated to aKG + aspartate.

OAA + glutamate -> glu/OAA transaminase -> aspartate + aKG

these can leave the mitochondria through antiporters. asp/glu antiporter.

this gives 3/2.5 atp per nadh

Front 74

why are there classical numbers and new numbers for ATP generation from NADH and FADH2

Back 74

because classical numbers do not take into account that in order to get ATP out of the mitochondria, it brings ADP in. the difference in charges takes away from some of the electrochemical gradient made for the protons.

Front 75

3 hypotheses of the coupling between oxidative respiration and phosphrylation

Back 75

1) chemical hypothesis (kind of like glycolysis)

NADH makes high phosphoryl-transfer substrate which then transfers to ADP

worked with the uncoupling - simply hyrdorlyze the high energy intermediate

problems: no evidence of this intermediate, also doesnt explain why mitochondrial membrane needs to be intact.

2) conformational hypothesis

a protein can change conformation to a higher energy state and then when it goes back down, it phosphrylates ADP. can be seen in muscles with actomyosin working backwards to create ATP.

problem was no way to uncouple!

3) chemiosmotic theory. accepted.

Front 76

myristate

Back 76

C14

Front 77

palmitate

Back 77

C16

Front 78

stearate

Back 78

C18

Front 79

oleate

Back 79

18:1

Front 80

linoleate

Back 80

18:2

Front 81

linolinate

Back 81

18:3

Front 82

arachidonate

Back 82

20:4

precurosr for prostaglandins and leukotrines

Front 83

what should the pH difference between inner and outer mitochondria be, what is it actually?

Back 83

should be about 3. but it is only 1. the reason is because there is also an electrical gradient which makes the overall gradient much larger.

Front 84

how could an ETC uncoupler work mechanistically?

Back 84

is a weak acid (bind protons) and
lipophilic (dissolves in membrane in both forms with or without the ion).

this then spreads the charge across the membrane and destroys the gradient.

Front 85

how can you prove chemiosmotic hypothesis?

Back 85

do a pH test. the external hsould acidify and internal alkylate!

use purple membrane bacterium (purple membrane and Fo and F1). light activates a proton pump and then ATP is synthesized!

Front 86

ionophores

Back 86

lipid soluble boats that carry ions.

they can destroy a membrane potential and uncouple the ETC

Front 87

validomycin

Back 87

an ionophore: C-O boat that can hold a K ion and carry across the membrane and release.

The gradient is then destroyed

Front 88

can a fatty acid be an uncoupler?

Back 88

no because when it flips into the mitochondria, the low pH makes it an anion and it cannot flip back.

uncouplers need to be able to go both ways.

Front 89

UCPs and their mechanisms

Back 89

uncoupling proteins that uncouple the mitochondria

UCP 1: a fatty acid anion carrier that helps the anion flip back to the cytoplasm. [done in brown fat so that there is uncoupled respiration producing heat]

UCP 2 and 3: not clear

Front 90

brown fat

Back 90

mice and young humans have this which has alot of mitochondria and respiration but it is uncoupled by UCP 1 so produces heat. [adult humans just shiver]

Front 91

difference between an ionophore and an uncoupler

Back 91

uncoupler: a weak acid that can carry protons to destroy both electrical and chemical gradient

ionophore: something that transports an ion (not necessarily H) can destroy electrical potential but not necessarily chemical.

Front 92

ATPase in the mitochondria

Back 92

Fo is the stalk (pore in which protons go through) and F1 is the ball.

F1 has the ATP synthesizing capacity. it turns like a motor when H goes down against its gradient.

Front 93

oligomycin

Back 93

chemical that inhibits phosphorylation of ATP in mitochondria by block the Fo pore (like a cork)

Front 94

how to get ATP from inside mitochondria to cytosol?

Back 94

use adenine nucleotide translocase.

shuttle ADP in and ATP out.

Front 95

atractiloside and bangkrekic acid

Back 95

adenosine nucleotide translocase inhibitor. now ATP cannot leave and ADP cannot come in.

in other words, it is an indirect phosphorylation inhibitor

Front 96

sources of Acetyl CoA

Back 96

fatty acids and glucose

Front 97

triglyceride

Back 97

glycerol with three fatty acids on it.

usually 1 and 3 positions are saturated and 2 position is unsaturated

Front 98

long chain fatty acids

Back 98

LChain: at least 14 Carbons

Front 99

regulation of triglyceride breakdown

Back 99

lipase is sensitive to phosphorylation by PKA. increases its activity 2-3 fold.

also the lipid droplets have a protein called paralipin which keeps lipase away. PKA also phosphorylates the paralipin to get it out of the way and allow lipase to come in and work (increases more then 2-3 fold)

ATGL - adipocyte triglyceride lipase

Front 100

how can you get glucose from fat?

Back 100

triglyceride breaks down into glycerol and fatty acids. the glycerol and become DHAP and through gluconeogenesis, become glucose

Front 101

for fatty acid oxidation, what pulls the reaction

Back 101

the PPi gets broken down into 2Pi by pyrophosphatase. that pulls the reaction forward

Front 102

where does beta oxidation occur? how does fatty acid get there?

Back 102

in mitochondria. fatty acid must be transferred to carnitine (CPT1) and then through the translocase into the mitochondria and made back into acyl-CoA (CPT2)

Front 103

CPT 1 regulation

Back 103

inhibited by malonyl CoA (acetyl CoA + COO-) - an enzyme used for FA synthesis

makes sense that if you want synthesis you should shut down oxidation

Front 104

regulation of FA synthesis

Back 104

AMP (low cellular energy state) activates AMPK.

AMPK (among other things) phosphorylates (and inhibits) Acetyl CoA Carboxylase which makes malonyl CoA.

Malonyl CoA is a substrate for synthesis and inhibits catabolism (blocks CPT 1).

so AMPK effectively shuts off synthesis and promotes the breakdown.

Front 105

If FA can flip across the membrane why do we need a transport?

Back 105

long chain FA's cannot be converted into CoA in the mitochondria, they must go through the transport system. this allows for the tight regulation of CPT1

note that medium and short chain fatty acids are free to enter mitochondria and can get CoA. (they do not depend on CPT1)

Front 106

why do you get more energy from fat then glucose

Back 106

fats are more reduced so more potential for taking off reducing equivalents (glucose has lots of oxygen already)

also C16 is more then C6

so fat is more dense

glycogen storage is hydrated (3x more water weight wise) whereas FA dont like water!

Front 107

knoops experiment

Back 107

experimented to find that beta oxidation occurs in pairs.

attached phenol to end of fatty acid chain and check urine.

if odd: get benzoic acid
if even: get benzoic methyl acid

Front 108

story of bumblebee

Back 108

bumblebee can fly when cold because it has an unregulated F1,6 bisphosphatase that can continually hydrolyze phosphates and generate heat.

Front 109

why is alanine such a large constituent of amino acids released from muscle cells? (like 50%)

Back 109

Not because muscle proteins have lots of alanine...

really because alanine is created from pyruvate and released to the liver to undergo gluconeogenesis. it is a carrier of energy.

Front 110

why is glycogen branching good?

Back 110

because allows for more ends to be attacked and glucose to be released at a faster rate when needed quickly.

Front 111

what happens to G6P from glycogenolysis in muscle and liver?

Back 111

muscle (needs energy) G6P goes to glycolysis

liver (releases energy) G6P goes to glucose and released.

Front 112

glycogen phosphorylase:

Keq?

Back 112

enzyme that uses inorganic P to hydrolyze glycogen and release G1P.

Keq is actually in reverse (1/3.6) but the high [Pi] allows the forward reaction to take place in vivo

Front 113

nucleoside diphosphate kinase

Back 113

a nonspecific enzyme that equilibriates di and triphosphate molecules.

for instance, you can get UTP from UDP by transfer from ATP...

Front 114

branching enzyme

Back 114

takes a long line of glycogen and makes a branch (at least 4 residues away) in a 1,6 branch.

Front 115

glycogen phosphorylase regulation

Back 115

A (tetramer): phosphorylated version (by phosphorylase B kinase)

B (dimer): inhibited by ATP and G6P but activated by AMP (lots)

PKA increases and partly activates phosphorylase b kinase. the new kinase turns B to A and strongly activates glycogenolysis.

ALSO: Ca released from muscle contraction can partly activate phosphorylase b kinase.

so Ca and PKA working together result in a strong activation of phosphorylase B kinase and glycogenolysis.

Front 116

glycogen synthase activation

Back 116

A (active) and B (less active) forms.

For B to be active it needs HIGH G6P.

B gets phosphorylated by PKA or GSK (unlike A in glycogen phosphorylase) and inactivated so that synthesis does not occur during starvation.

Front 117

PKA effects

Back 117

- activates phosphorylase kinase B; leading to the activation of glycogen phosphorylase (A) and glycogenolysis

- inactivates glycogen synthase (phosphorylates it to B) to stop glycogen synthesis

- phosphorylates pyruvate kinase in the liver to deactivate it and stop glycolysis.

- phosphorylates PFK II/ FBPase II complex to FBPaseII to inhibit glycolysis and stimulate gluconeogenesis

Front 118

where are glucagons effects and epinephrines effects?

Back 118

liver: glucagon triggers cascade; epi triggers the release of calcium

muscle: epinephrine triggers cascade; glucagon has no effects

Front 119

how does coffee help you be alert?

Back 119

it is a methyl xanthine that inhibits the phosphodiesterase that converts cAMP to AMP and thus potentiates the PKA signaling which is to increase overall energy level of the body.

Front 120

Hers Disease

Back 120

missing glycogen phosphorylase in liver.

now cannot break down glycogen there so gluconeogenesis is amped up.

so can survive

Front 121

Von Gierke Disease

Back 121

missing G6Phosphatase

Big problem because you cannot get glucose out to feed the body either through glycogenolysis or gluconeogenesis.

Real problem. can stand only limited starvation

Front 122

McArdle Disease

Back 122

missing glycogen phosphrylase in muscle.

so muscles are easily fatigued during exercise

Front 123

Eye-strain disease

Back 123

missing glycogen phosphorylase b kinase.

so they cannot convert phosphorylase B -> A.

but AMP can still activate phosphrylase B

and Ca can still partly activate phosphorylase b kinase

Front 124

how to measure activity of the pentose phosphate pathway

Back 124

radiolabel C1 and see how much radiolabel is given off as CO2.

Front 125

PRPP

Back 125

5-phosphoribosyl-1-pyrophosphate

an activated ribose-5-phosphate which serves as the site for each
addition of each of the atoms of the purine ring

is also used as a base to attach the pyrimidine ring in pyrimidine synthesis.

Front 126

AICAR

Back 126

"ZMP" looks like adenylate but has an imidazole and activates AMPK.

Front 127

purine synthesis regulation

Back 127

both PRPP synthetase and amidotransferase are feedback inhibited by IMP, AMP, GMP (and diphosphates).

ATP, GTP inhibit the amidotransferase

AMP inhibits adenylosuccinate synthetase; ATP stimulates IMP D.H.

GMP inhibits IMP D.H.; GTP stimulates adenylosuccinate synthetase

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importance of nucleotides

Back 128

- DNA and RNA
- energy (ATP and GTP)
- can activate sugars (UDP-glucose)
- lipid synthesis (CDP-diacylglycerol)
- component of coenzymes (cAMP)
- phosphate donors
- signaling

Front 129

Pyrimidine synthesis summary

Back 129

involves two precursors (carbamoyl phosphate and aspartate) followed by ring closure to form a base (orotate), which is then added
to PRPP (in a reaction which resembles nucleotide salvage) to form a nucleotide.

Front 130

Pyrimidine synthesis regulation

Back 130

First reaction (carbamoyl phosphate
synthetase) is inhibited by pyrimidine nucleotides (UTP, UDP, UMP, CTP) and activated by ATP.

Second reaction (aspartate transcarbamoylase) is also inhibited
by CTP and activated by ATP.

Front 131

ribonucleotide reductase regulation

Back 131

one enzyme for all 4 types of DNA. but need to make sure that there are equal amounts of all 4 types.

allosteric sites can regulate substrate specificty

dATP promotes UDP and CDP reduction but reduces OVERALL activity of enzyme.

dTTP promotes GDP and inhibits UDP and CDP

dGTP promotes ADP

regulators are triphosphates and substrates are diphosphates.

Front 132

how to get dUMP?

Back 132

must go from dUDP -> dUTP (nucleotide diphosphate kinase) and then special dUTPase (dUTP -> dUMP).

or deaminate dCMP.

Front 133

flouro-uracil

Back 133

a form of chemotherapy.

converted to flouro-deoxyuridylate and a suicide inhibitor of thymidylate synthase

Front 134

methotrexate (aminopterin or amethopterin)

Back 134

a form of chemotherapy:

folate analogues that inhibit dihydrofolate reductase, thereby
preventing the resynthesis of tetrahydrofolate and stopping thymidine synthesis and blocking DNA synthesis and division...

Front 135

Gout (and how to treat)

Back 135

results from increased formation or
decreased excretion of uric acid.
Major cause is excessive purine synthesis leading to elevated levels of urate in serum and urine and deposits of urate crystals in joints of the extremities and kidney.

Treatment utilizes the drug allopurinol, a base analogue of hypoxanthine, which inhibits the
enzyme xanthine oxidase (suicide inhibition) and stops production of urate.

Front 136

Lesch-Nyhan syndrome

Back 136

complete absence of HGPRT;

results in a buildup of PRPP and denovo purine synthesis.

symptoms are gout (due to excessive urate production, mental retardation, and self-mutilation (due to the build-up of guanine and hypoxanthine).

Front 137

Orotic acidurea

Back 137

a deficiency in pyrimidine synthesis resulting in orotate in the urine

Front 138

severe combined immunodeficiency
disease (SCID)

Back 138

Adenosine deaminase deficiency which results in a massive buildup of dATP which inhibits ribonucleotide reductase and stops DNA synthesis.

suffers from immunodeficiency

Front 139

why is ammonia a toxin?

Back 139

most is in protonated state (ammonium) but ammonia can attack as a solid nucleophile to displace esters and create amides and cause major changes.

ammonium itslef also interacts with a receptor (N-methyl-D-aspartate receptor) in neurons and causes damage

Front 140

amino acids that can be transaminated to pyruvate

Back 140

alanine, glycine, serine, cysteine

Front 141

AAs that can be transaminated to OAA

Back 141

aspartate, asparagine

Front 142

dynamic state of body proteins

Back 142

levels of body proteins are very plastic and proteins are continually degraded and synthesized (most do not last that long). this lends to a dynamic state.

Front 143

Ebb and Flow of AA Nitrogen

Back 143

AA/protein from diet -> AA pool.

AA pool can make body proteins and other nitrogenous compounds.

After all the needs are met, the rest of the AA are metabolized and N is made into urea (non-toxic) and the alpha keto acid is used as metabolic intermediates.

Front 144

glycogenic AA vs ketogenic AA

Back 144

glycogenic can have ketoacids made into glucose metabolite [i.e. pyruvate, OAA, succinyl CoA, alpha KGA, fumerate]

ketogenic can have keto acid made into a ketone body (acetoacetate, b-hydroxybuterate) for fuel for the brain

Front 145

non-essential AAs

Back 145

non-essential if the keetoacid skeleton is readily available

gly, ala, ser, cys (pyruvate)

asp, asn (OAA)

gln, glu (alpha KGA)

proline (from glu)

tyrosine (special)

Front 146

essential AAs

Back 146

AA whose carbon skeletons are not available must be gotten from diet

thr, met, val, leu, ile, his, lys, arg, phe, trp

Front 147

how is tyrosine made?

Back 147

from phenylalanine.

enzyme called phe hydroxylase. uses a mole of oxygen and releases water.

the water must be reduced from something else: tetrahydrobiopterin (THBT) gets oxidzed to DHBT and oxygen becomes reduced.

DHBT reductase reduces DHBT to THBT. gets reducing electrons from NADPH.

phe -> tyrosine

Front 148

what happens if you dont have phe hydroxylase?

Back 148

can get disease called PKU (phenylketonuria).

phe accumulates and tyr is not synthesized.

phe can be converted to phenylpyruvate, phenylacetate, phenylacetyl-gln...

develop neurological problems very quickly. myelin sheath gets damaged

not clear which by-product is the most toxic

can also get PKU if DHBT reductase does not work.

treat with low Phe diet

Front 149

what happens if DHBT reductase is missing?

Back 149

can develop PKU. also cannot make 5HT so much more serious problems as well.

Front 150

kwashnikov

Back 150

disease where missing lys from diet. serious problems occur with growth and cns.

Front 151

nitrogen balance measurement

Back 151

a way to see if the body is healthy.

equilibrium: N in = N out: healthy with balanced diet

N in > N out: New tissue deposition as in growth, recovery from injury or surgery, pregnancy

N out > N in: Wasting diseases (i.e., cancer), starvation, dietary lack of one or more essential
amino acids, or aging

Front 152

where does PLP come from

Back 152

vitamin B6. oxidized and then phosphorylated

Front 153

deficiency of alanine, and excess of aspartate. how can you compensate?

Back 153

asp + alpha-KGA -> OAA + glu

and then, take glu and react it with a pyruvate to get a-KGA and alanine!

so can make up for any deficiency by redistributing through transamination.

Front 154

mechanism of transaminases

Back 154

PLP is in active site attached to E amino group of a lys residue in a schiff base conformation.

the amino group of the AA displaced the amino group of lys and PLP forms schiff base with AA. releases water (go from enzyme aldimine to substrate aldimine)

schiff base forms quinoid intermediate. h-bond between N and O keeps structure planar and stabilized.

quinoid makes carbanion and then water comes and displaces the new keto acid leaving the PMP behind.

the last step is pretty much the same reaction in reverse. (the new keto acid comes in and electrons push and out pops the new AA and PLP.

Front 155

release and temporary storage of ammonia

Back 155

ammonia can be released from glu, his, thr, ser.

glu is through oxidative deamination
glu + nad (or nadp) + h20 -> a-KGA + nh3 + nad(p)h + h (glu dehydrogenase)

can actually get energy! this enzyme is allosterically regulated by energy state of mitochondria (AMP activates ATP inhibits)

the other three are not oxidative. simply called direct deamination.

ser/thr (ser or thr dehydratase)
his (his lyase)
ammonia can be stored in glu and asp to make gln or asn by a synthetase (gln synthetase) and reverse rxn catalyzed by amidases (i.e. glutaminase)

Front 156

why do you need energy to put NH3 on glu? (to make gln)

Back 156

because the carbon in glu is slightly negative and cannot accept the NH3 attack. so phosphate comes in to stablize the carbon and allow the attack by the nh3

Front 157

definition of lipid

Back 157

something that dissolves in fat and not water

Front 158

functions of lipids (7)

Back 158

- compartmentalization

- energy storage

- signaling

- hormones

- protein fxn and localization

- receptors

- anti-oxidants

Front 159

why don't fatty acids make bi layers?

Back 159

because they are more soluble in water then phospholipids and thus cannot make bilayers.

Front 160

functions of myristate

Back 160

- < 1% of FA composition
- covalent modification of proteins that is necessary for function (used for targeting and subcellular localization)

- added to N- glycine

- added co-translationally

- enzyme that myristolates is called N-myristoyl-Transferase

- i.e. G-1-alpha protein subunit

Front 161

functions of palmitate

Back 161

- major FA in the body

- protein modification of cys/thr thioester linkages

- post-translational

- reversible

- palmitoyltransferase is enzyme. (not specific)

- important for G protein fucntion (without it, G-gamma subunit does not work as well)

- i.e. caveolin (a scaffolding protein)

- not necessarily to target to membrane...not quite sure what the function is...probably protein interactions

Front 162

functions of stearate

Back 162

we make it.

structural fatty acid.

Front 163

functions of palmitoleate

Back 163

structural

Front 164

functions of linoleate

Back 164

we cannot make this.

it is essential (get from seed oils).

can be delivered through the skin

Front 165

functions of arachidonate

Back 165

made from linoleate (essential)

precursor for prostaglandins and leukotrines...biologically important FA derivatives.

Front 166

fish oils

Back 166

"omega 3's"

prevent heart problems. can stop some bad processes from arachidonate (like bad thromboxines)

Front 167

what happens if free fatty acid levels go up in cell

Back 167

toxic and disrupts membranes (makes them leaky)

Front 168

what are FAs complexed to in blood

what about in cell?

Back 168

albumins.

in cell: complexed to cholesterol, glycerol (triglyceride)

Front 169

what does FA-cholesterol complex do?

Back 169

stops bad effects of too much cholesterol by removing it from the membrane

Front 170

triglycerides

Back 170

sn1 is usually saturated
sn2 usually unsaturated
sn3 could be phospho

Front 171

zwitteron

Back 171

one molecule with two differnt charges on it. (like phosphatidylcholine with positive choline and negative phopshate)

has influence on chemical nature

Front 172

why have so many different kinds of head groups and fatty acids for bilayer?

Back 172

to help you mix and match structurally.

also different tissues have different compositions

Front 173

cardiolypin

Back 173

phospholipid group that is found mainly in the mitochondria

Front 174

outer membrane vs inner membrane composition

Back 174

outer: PC and sphingomyelin and glycolipids

inner: PE (ethanolamine)and PS (serine)

important to maintain it. uses flipase enzyme

Front 175

if PS is on outer what happens?

Back 175

signals apoptosis

happens as cell ages and less ATP and less glycolysis. flippases become less active...

Front 176

physical characteristics of membrane and how they are controlled

Back 176

permeability: want it to be impermeable to anything that is charged (need channels) but is permeable to nonpolar molecules

fluidity: (hand in hand with permeability).

fluid mosaic model

controlled by: FA composition and cholesterol content (van deer wall allows for good movement without covalent bonds)

phospholipid composition (not so imp)

Front 177

FRAP

Back 177

flourescence recovery after photobleaching.

used to measure fluidity of membranes

Front 178

FA composition rules for fluidity

Back 178

want more fluidity (lower Tm): use shorter chains and more double bonds

Front 179

cholesterol rules for fluidity

Back 179

want more fluidity, use more cholesterol.

but not too much (at really high concentrations they come out of solution)

Front 180

digitonin

Back 180

looks like cholesterol. causes large pores in membranes. cytosol comes out.

so can selectively determine where a protein is by using increasing amounts of digitonin and different membranes have different thresholds (different amounts of cholesterol)

Front 181

why is energy stored as fat and not glycogen

Back 181

more dense (more calories/gram)

more fluid and flexible (not a sugar cube)

Front 182

what happens to FA in cell

Back 182

gets immediately activated into CoA-FA to be made into a TG

Front 183

ACP

Back 183

a domain in the FA synthase complex in mammals but an enzyme in bacteria that activates FA into FA-CoA

Front 184

where are triglycerides made?

Back 184

in all cells. but mostly adipose and liver.

liver packages it into LDL and ships it out--usually to adipose

Front 185

xenical

Back 185

pancreatic lipase inhibitor

for diets. stops fat absorption.

problem is that you get steatorhea (can become incontinent)

dont eat alot of fat!

Front 186

ollestra

Back 186

diet drug.

can prevent vitamin absorption.

OH groups have FA attached and pancreatic lipase cannot digest and so it excretes.

can still get steatorhea

Front 187

can you radiolabel CO2 in FA biosynthesis and have it in the product?

Back 187

no it is not in the end product

Front 188

palmitate modifications

Back 188

add a malonyl CoA to elongate. (elongase)

desaturase adds a double bond using ETC. very specific

Front 189

sterculic acid

Back 189

desaturase inhibitor

used alot in cattle to make meat perfect

Front 190

ketone bodies

how are they used

problems

Back 190

high fat, no carb diet.

OAA levels drop because of gluconeogenesis and Acetyl CoA cannot do anything...

3 acetyl CoA --> acetoacetate --> beta hydroxy buterate and acetyl Coa

b-OH-butyrate can go into blood and be used in many cells very well even brain.

b-OH-butyrate --> acetoacetate --> 2 acetyl CoA (uses special enzyme not in liver)

problem is they are acids. and can cause ketosis - especially in diabetics. low glucose = high lipolysis = high ketone bodies...acidosis

Front 191

metabolic syndrome

how is it linked to cardio problems

Back 191

common denominator of all diseases pertaining to metabolism problems

develop insulin resistance and semi diabetes...

elevated glucose serum levels and LDL (bad loading cholesterol into tissues)

decreased HDL (good...clearing cholesterol)

fatty acids are very toxic to cardiomyoctyes

Front 192

purpose of paper

Back 192

to find the mechanism through which fatty acids are toxic to cells

Front 193

how to find out what are factors in cytotoxicity of a fatty acid if you dont know the pathway

Back 193

find mutants that do not die and then do protein assays to find out why

Front 194

CHO cells

Back 194

chinese hamster ovary cells

Front 195

forward genetics

Back 195

isolating mutants to figure out what gene you knocked out

Front 196

how to isolate mutants

Back 196

first use mutagens to increase frequency of mutations (pROSAgeo)

then select for whatever you want (treat all the mutants with palmitate that would normally kill and isolate the mutants that dont die)

- problem is that each one might be resistant for a different reason...so take a single cell and replicate it so that you get a pure cell line with the same mutation

Front 197

mutagen used in paper

Back 197

a retrovirus.

goes into cell and DNA is made off of RNA and the DNA is very good at inserting into the genome. inserts randomly into the chromosomes.

in some places it wont do anything.

the DNA encodes for Bgeo gene that infers resistence to G418.

This helps select for only the cells that were transfected with the retrovirus with the virus actually expressed,

has to get in the DNA and a functional gene that it will disrupt and in its place will make Bgeo.

Front 198

limitations of the mutagen used

Back 198

some genes that are important for the study are also important in the cell and if knocked out it will die. so some genes will be missed.

also since it is a diploid ovary cells, you need the insertion to be in both alleles! luckily though, some CHO cells are semi-haploid (one allele is not expressed well)

Front 199

once you have selected your mutants, how do you find the gene that was disrupted?

Back 199

use 5' race.

a way of obtaining unknown sequences.

know the viral sequence. design a primer complementary to the virus and use reverse transcriptase to generate a sequence that copies viral AND unknown region.

then chew up with RNAse and then left with DNA.

can add TdT C to the 3' end and make a tag and then run through PCR and now you have the gene to sequence

Front 200

where was the viral DNA translocated in the eEF1A gene?

Back 200

in the 5' UTR (which is more homologous then the 3' UTR evolutionarily)

Front 201

purpose of propitidium iodide staining

Back 201

it stains dead cells and it is used to make sure that the mutant is only resistant to palmitate and not some other lethal drug like actinoycin D...

shows that it is selective

Front 202

TUNEL

Back 202

looks for apoptotic factors...DNA fragments.

if there are ends then the cell is probably going through apoptosis.

Front 203

DCF Flourescence

Back 203

interacts with reactive oxygen species in cell (that occurs during cell death).

if DCF floourescence goes up with increased palmitate, then the cell is releasing more oxidative species and leading towards death

Mean DCF fluorescence is indicative of relative cellular ROS level and can be measured by flow cytometry

Front 204

problems with the paper

Back 204

lethal mutations cannot be analyzed

also the diploid nature makes it hard too