BIO 311 Exam #1

allosteric regulation

• bind to the enzyme → conformational change → decrease enzyme activity

• feedback inhibition: regulate levels of the synthesized end product

• reversible

covalent modification

• add phosphate groups → add negative charge and change electrostatic properties

• reversible

proteolytic modification

• digestive enzymes, clotting factors

• synthesized as inactive pro-enzymes, cut up, segments activated

• not reversible

catabolism

• degradation

• release energy

• creates ATP, NADH, NADPH, FADH2

• exergonic processes

anabolism

• synthesis

• uses energy

• ATP, NADH, NADPH, FADH2

• endergonic processes

heterotroph

• use C from food

• energy from degradation

• makes CO2, H2O

autotroph

• use C from air (CO2)

• energy from sunlight

• make O2, H2O

oxidation

• loss of electrons

• losing electrons = reducing agent

• catabolism

reduction

• gaining electrons

• gaining electrons = oxidizing agent

• anabolism

FAD/FADH2 and FMN/FMNH2

• act as coenzymes in many enzyme-catalyzed RedOx reactions

• can accept 1 or 2 hydrogens

redox common in catabolism

1. substrate undergoes oxidation

   1. lose 2H (2 p+ and 2 e-)

2. oxidized form of NAD accepts hydride

   1. :H- (1 p+, 2e-)

redox common in anabolism

1. NADH donates hydride to oxidized substrate

2. substrate becomes reduced

lactate → pyruvate

• lactate dehydrogenase

• 2e- and 2H+ removed from C2 of lactate (alcohol) to make pyruvate (ketone)

• reversible

homolytic cleavage

each atom keeps one of the bonding electrons

heterolytic cleavage

one of the atoms keeps both of the bonding electrons

isomerization

• redistribution of electrons within a molecule

• can result in isomerization, transposition of double bonds, cis-trans rearrangement

• isomerases

eliminations

• elimination of water

• introduce C=C between 2 saturated Cs

acyl group transfer

addition of nucleophile to carbonyl C to form a tetrahedral intermediate

phosphoryl group transfer

attachment of goof LVG to metabolic intermediate to “activate” the intermediate for subsequent reactions

not simple ATP hydrolysis

1. formation of phospho-substrate intermediate gives greater free energy

2. displacement of P group from substrate

glycolysis

• cytoplasm

• oxidation of glucose → pyruvate

• creates ATP, NADH

TCA cycle

• mitochondria

• pyruvate → citrate

• creates NADH, FADH2, CO2

electron transfer & oxidative phosphorylation

• mitochondria

• p+ pumps and e- transfer drive ATP synthesis

• creates ATP, H2O

sucrose

• 1,2-linked α-glucose + β-fructose

• α-1,2-glycosidic linkage

• source: sugar cane

lactose

• 1,4-linked β-galactose + α-glucose

• β-1,4-glycosidic linkage

• source: milk

maltose

• 1,4-linked α-glucose + α-glucose

• α-1,4-linked glycosidic bond

• source: hydrolyzed starch

why can’t we digest cellulose?

• humans do not have the enzymes that can break β-1,4-glycosidic bonds

• cellulose is too large for lactase to accommodate in its active site

amylopectin

more branching, α-1,4-linked glucose with α-1,6-linkages at branch points

amylose

long, unbranched chains of D-glucose; α-1,4 linked

glycogenin reactions

• attachment of UDP-glucose to glycogenin protein

• transfer of glucosyl residues to existing (glu)n-glycogenin

glycogen breakdown steps

1. (glycogen)_{n glu} + P_i → (glycogen)_{n-1 glu} + G1P

2. G1P → G6P

3. G6P + H₂O → glucose + P_i

4. removal of branch points

glycogen breakdown - enzymes

1. glycogen phosphorylase

2. phosphoglucomutase

3. glucose-6-phosphatase

4. debranching enzyme

reducing sugars

sugars that reduce mild oxidizing agents

debranching enzyme

• remove all but one glucosyl residue from a branch point and transplants the short chain to neighboring branch

• α-1,6-glucosidase: removes last glucosyl residue as glucose

glycogen synthase

(glycogen)_{n glu} + UDPG → (glycogen)_{n+1 glu} + UDP

glycogen synthesis

1. glucose + ATP → G6P + ADP

2. G6P → G1P

3. UTP + G1P → UDP-glucose + pyrophospate

4. UDP-glu + (glycogen)_{n glu} → (glycogen)_{n+1 glu} + UDP

5. pyrophosphate + H₂O → 2P_i + H⁺

6. branching enzyme

glycogen synthesis enzymes

1. hexokinase (glucokinase in the liver)

2. phosphoglucomutase

3. UDP-glucose pyrophosphorylase

4. pyrophosphatase

5. branching enzyme

branching enzyme

• glycogen synthase makes α-1,4 glycosidic bonds

• branching enzyme transplants short chain to introduce branch by forming an α-1,6-glycosidic bond

insulin

• lowers blood glucose

• inhibits glycogen breakdown (promotes synthesis)

• inhibits gluconeogenesis

epinephrine and glucagon

• raise blood glucose

• stimulate glycogen breakdown

• stimulate gluconeogenesis

low blood glucose: increased glycogen breakdown

1. low blood glu

2. inc glucagon

3. inc cAMP

4. inc PKA

5. PKA phos + activates phosphorylase kinase

6. inc phosphorylase kinase phos + activates glycogen phosphorylase

7. inc glycogen phosphorylase

low blood glucose: decreased glycogen synthesis

1. low blood glu

2. inc glucagon

3. inc cAMP

4. inc PKA phos + deactivates glycogen synthase

5. dec glycogen synthase

high blood glucose: decreased glycogen breakdown

1. inc insulin

2. inc insulin-sensitive protein kinase

3. inc PP1

4. PP1 dephos + inactivates phosphorylase kinase

5. dec phosphorylase kinase leads to dec glycogen phosphorylase (inactive)

high blood glucose: increased glycogen synthesis

1. inc insulin

2. inc PKB

3. PKB phos + inactivates GSK3

4. inactive GSK3 leads to dephosphorylated glycogen synthase (active)

5. more glycogen synthase

allosteric effects: ATP

• G6P

• high E state

• inhibit phosphorylase

allosteric effects: AMP

• activate phosphorylase

• E state of cell is low

• glycogen breakdown

insulin & G_M protein

• phosphorylation of GM site 1

• activate PP1

• dephosphorylates glycogen phosphorylase kinase, glycogen phosphorylase, and glycogen synthase

epinephrine & G_M protein

• phos of Gm site 2

• PP1 dissociates from glycogen

• prevent PP1 access to glycogen phosphorylase & glycogen synthase

products of glycolysis

per glucose molecule:

• 2 ATP

• 2 NADH

• 2 pyruvate

3 possible fates for pyruvate

1. aerobic respiration (CO2 + H2O)

2. anaerobic respiration (lactate)

3. anaerobic respiration (ethanol)

glycolysis reaction overall

glucose + 2NAD⁺ + 2ADP + 2P_i → 2 pyruvate + 2NADH + 2H⁺ + 2ATP + 2H₂O

glycolysis prep phase

• 4 steps

• converts 6C sugar to 2 3C sugars

• uses 2 ATP

glycolysis payoff phase

• 6 steps

• converts 2 3C sugars to 2 pyruvate

• makes 4 ATP (2 from each 3C sugar)

glycolysis step 1

glucose → G6P

• phosphorylation

• ATP → ADP

• hexokinase

• priming reaction

• rate limiting step

• “traps” glucose as G6P which does not diffuse out of cells or bind to glucose transporters

glycolysis step 2

G6P → F6P

• phosphohexose isomerase

• reversible

• isomerization

glycolysis step 3

F6P → F-1,6-BP

• PFK-1

• ATP → ADP

• phosphorylation

• not reversible

• first committed step

• rate-limiting step

• second priming step

glycolysis step 4

F-1,6-BP → DHAP + GAP

• aldolase

• reversible

• cleavage

glycolysis step 5

DHAP → GAP

• triose phosphate isomerase

• reversible

glycolysis step 6

GAP → 1,3-BPG

• GAP dehydrogenase

• reversible

• NAD⁺ → NADH + H⁺

• generation of a high-energy compound

glycolysis step 7

1,3-BPG → 3-phosphoglycerate

• phosphoglycerate kinase

• 2 ADP → 2 ATP

• reversible

• substrate-level phosphorylation

glycolysis step 8

3-PG → 2-PG

• phosphoglycerate mutase

• reversible

• rearrangement

glycolysis step 9

2-PG → PEP

• enolase

• reversible

• H₂O released

• generation of a high-energy compound

glycolysis step 10

PEP → pyruvate

• pyruvate kinase

• 2 ADP → 2 ATP

• not reversible

• rate-limiting

• substrate-level phosphorylation

fermantation

• lactic acid, ethanol

• energy extraction without consumption of oxygen

• no net change in the [NAD⁺] or [NADH]

lactic acid fermentation

pyruvate → L-lactate

• reversible

• lactate dehydrogenase

• NADH + H⁺ → NAD⁺

• 2 redox reactions; no net change in oxidation state of C in glucose

• no net change in oxidation

• 2ATP/glucose extracted in conversion of glucose → lactate

ethanol fermentation

pyruvate → acetaldehyde

• reversible

• CO2 released

• pyruvate decarboxylase

acetaldehyde → ethanol

• reversible

• NADH + H⁺ → NAD⁺

pasteur effect

yeast consume more sugar when grown under anaerobic conditions

hexokinase deficiency

• reduced glucose breakdown

• reduced ATP production

• reduced BPG production

• not as easy for Hb to assume T-state

pyruvate kinase deficiency

• reduced ATP production

• RBCs become deformed/lyse

• Hb carries less O2

• individuals with a deficiency in pyruvate kinase would be expected to display a decrease in hemoglobin affinity for oxygen 

glycolysis & cancer

• cancer cells grow more rapidly than blood vessels supplying them

• hypoxic tumors express HIF-1

• HIF-1 increases gene expression (glycolytic enzymes, GLUT)

• HIF-1 stimulates growth of vasculature (VEGF)

anaplerotic reaction

reactions that replenish intermediates depleted by other reactions

generation of acetyl-CoA

1. CoA gets 2C from pyruvate in the form of an acetyl group

2. High-energy thioester linkage

3. Multi-enzyme process (coupling)

E1

pyruvate dehydrogenase

• uses TPP as bound cofactor

• attacks C2 of pyruvate, releases CO2

• TPP remains bound to hydroxyethyl group

E2

dihydrolipoyl transacetylase

• lipoic acid: cofactor covalently bound to Lys of E2

• creates long, flexible arm - can move between active sites of all 3 PDH enzymes

• disulfide reduced to SH + SH

• lipoamide side chain extends to E1

• transfers hydroxyethyl from TPP to dihydrolipoamide

• partial reduction creates acetyl group

• second reduction transfers acetyl group to CoA

E3

dihydrolipoyl dehydrogenase

• resets the system

• catalyzes the regeneration of disulfide (oxidized) form of lipoamide/lipolysine

  • uses bound cofactor (FAD)

• NAD+ oxidizes FADH₂ to regenerate FAD

  • NAD becomes reduced (NADH + H+)

• PDH complex regenerated; NADH + H+ made

acetyl-CoA derived from FA

1. creating a fatty acyl-CoA

   1. thiol group of CoA-SH carries out a nucleophilic attack

2. β-oxidation

   1. each 4-step “pass” removes one acetyl group (2C) from chain to make acetyl-CoA

   2. also makes FADH2 and NADH/H+

TCA cycle step 1

acetyl-CoA + oxaloacetate → citrate

• citrate synthase

• H2O → CoA-SH

• condensation

• rate-limiting

• not reversible

TCA cycle step 2

citrate → cis-aconitate

• aconitase

• reversible

• dehydration (H2O released)

TCA cycle step 3

cis-aconitate → isocitrate

• aconitase

• reversible

• hydration (H2O put in)

TCA cycle step 4

isocitrate → α-ketoglutarate

• isocitrate dehydrogenase

• rate-limiting

• not reversible

• oxidative decarboxylation (CO2 released)

• NADH released

• manganese in the enzyme active site helps to stabilize the intermediate

TCA cycle step 5

α-ketoglutarate → succinyl-CoA

• α-ketoglutarate dehydrogenase

• rate-limiting

• not reversible

• CoA-SH → CO2

• oxidative decarboxylation

• NADH released

• TPP, NAD, FAD co-factors

TCA cycle step 6

succinyl-CoA → succinate

• succinyl-CoA synthetase

• reversible

• GDP/ADP → GTP/ATP

• CoA-SH released

• substrate-level phosphorylation

TCA cycle step 7

succinate → fumarate

• succinate dehydrogenase

• reversible

• FADH2 released

• dehydrogenation

• electrons flow from FAD→Fe/S→ETC

TCA cycle step 8

fumarate → malate

• fumarase

• reversible

• hydration (H2O put in)

TCA cycle step 9

malate → oxaloacetate

• malate dehydrogenase

• NADH released

• dehydrogenation

• reversible

pyruvate → acetyl-CoA

• inhibited by ATP, acetyl-CoA, NADH, fatty acids

• activated by AMP, CoA, NAD+, Ca2+

acetyl-CoA → citrate

• inhibited by NADH, succinyl-CoA, citrate, ATP

• activated by AMP

isocitrate → α-ketoglutarate

• inhibited by ATP, NADH

• activated by Ca2+, ADP

α-ketoglutarate → succinyl-CoA

• inhibited by succinyl-CoA, NADH

• activated by Ca2+

what does coupling depend on?

• sequential redox reactions that pass e- from NADH to O2

• compartmentalization of these reactions within the mitochondrion

• generation of proton gradient

2 ways ATP is produed

1. substrate level phosphorylation

2. oxidative phosphorylation

electron transport

• e- carried by reduced co-enzymes are passed through chain of proteins and coenzymes

• drives generation of proton gradient across inner mitochondrial membrane

oxidative phosphorylation

proton gradient runs downhill to drive synthesis of ATP

standard reduction potential

• Eº’

• measure of how easily a compound can be reduced

• more positive = the more the compound “wants” electrons

complex I prosthetic groups

FMN, Fe-S

complex II prosthetic groups

FAD, Fe-S

complex III prosthetic groups

Heme, Fe-S

complex IV prosthetic groups

Hemes; Cu_A, Cu_B

ubiquinone

• lipid-soluble carrier molecule

• CoQ, or Q

• lives/moves in mitochondrial membrane

• complete reduction requires 2e- and 2p+ (get them from matrix)

• shuttle e- from complex I and II → III

complex I

NADH-dehydrogenase

• FMN and Fe-S centers

• electron flow: NADH — FMN, FMNH2— Fe3+, Fe3+ — Fe2+

• e- ultimately shuttled to Q

• energy of electron transfer used to pump 4H+