Glycolysis and TCA Cycle - Abali 2/26/16

Question 1

glycolysis overview

Answer 1

• breakdown of glucose to get energy (ATP)
• conducted by all tissues

all cells pick glucose up via glucose transporters

step 1 from there: phosphorylation of glucose via HEXOKINASE or GLUCOKINASE

Question 2

types of glucose transporters

Answer 2

active vs facilitated transport

insulin sensitive vs. insulin insensitive

active transport [Na-glucose cotransporters]

• insulin-insensitive
  
  • intestinal epithelium
  • renal tubules
  • choroid plexus

facilitated transport [dependent on glucose conc gradient]

• insulin-sensitive [GLUT4]
  
  • sk muscle, adipose tissue (“need insulin 4 GLUT4 in the 4 muscle/fat limbs”)
• insulin-insensitive
  
  • most tissues [liver, brain, RBCs, etc]

Question 3

GLUT4 mobilization

Answer 3

insulin dependent

muscles/adipose tissue

in absence of insulin, GLUT4 is sequestered intracellularly in vesicles

​1. insulin binds to cell membrane receptor, upregulates recruitment of GLUT4 to cell membrane

2. GLUT4 increases insulin-mediated uptake of glucose into cell
   * glucose phosphorylated to keep it trapped in cell
3. when insulin drops, GLUT4 moves back into intracellular storage pool for recycling
   * vesicles fuse to form endosome

Question 4

key GLUT transporters

• relative Km
• site of action

Answer 4

GLUT1 : low Km (1)

• basal glucose uptake in RBCs and brain

GLUT2 : high Km (15-20)

• pancreatic beta cells (regulation of insulin)
• liver (storage of excess glucose)

GLUT3 : low Km (1)

• basal glucose uptake in brain neurons

GLUT4 : medium Km (5) - insulin dependent

• sk muscle, adipose tissue

Question 5

how does a cell hang on to glucose?

Answer 5

“tagging” with ATP makes glucose → glucose-6-phosphate

• G6P v hydrophilic, won’t diffuse out of cell
• catalyzed by glucokinase (liver) or hexokinase (all tissues)

Question 6

hexokinase vs glucokinase

• location
• relative Km
• inhibition

Answer 6

hexokinase (works at max levels even when glucose low)

• all tissues
• low Km
• G6P feedback inhibition

glucokinase (stores continuously, but best when glucose is high - insulin dep)

• liver
• high Km
• no feedback inhibition

implication: lowish Km of GLUT4 and low Km of hexokinase means muscle/fat are priority, but there is feedback inhibition to prevent them from trapping so much glucose that plasma stores drop

similarly, high Km of GLUT2 and high Km of glucokinase means liver only picks up glucose for storage when there’s enough to go around but lack of feedback inhibition means it can do this continuously (taking in more glucose over time)

Question 7

de vivo disease

Answer 7

hereditary deficiency of GLUT1

• drop in insulin-indep GLUT1 which picks up glucose in brain
  
  • decreased glucose in CSF

symptoms

• seizures and devpt delay
• neuro symptoms: ataxia, dystonia, dysarthia

tx

• ketogenic diet (high protein/fat) - ketones provide alt energy source for brain in absence of glucose

Question 8

glycolysis: two phases, three enzymes, net gains

Answer 8

two phases

1. “preparation”

6-carbon glucose → 3-carbon glyceraldehyde-3-P

2. “payoff”

G3P → pyruvate

three regulated enzymes (kinases)

1. gluco/hexokinase
2. phosphofructokinase-1 (PFK1)
3. pyruvate kinase

net gains

• 10 rxns that turn 6C glucose into 2 x 3C pyruvate
  
  • ​prep: 2ATP consumed
  • payoff: 4ATP + 2NADH produced
• ​most rxns are reversible (except for the ones regulated by the three enzymes)

Question 9

3 key regulated rxns of glycolysis

• regulation by hormones

Answer 9

in general

• insulin: upregulates glycolysis (fed state)
• glycogen: downregulates glycolysis (fasted state)

glucokinase: glucose → G6P

PFK1: fructose-6P → fructose-1,6-bisphosphate

pyruvate kinase: phosphoenolpyruvate → pyruvate

Question 10

regulation of hexokinase vs glucokinase

Answer 10

hexokinase

• feedback inhibition by G6P

glucokinase

• inhibited by F6P : gets GK transported into nucleus and sequestered there via binding to GKRP (GK reg protein)
  
  • inhibition reversed either by high intracell glucose or high intracell F1P
• expression of GK increased by insulin

Question 11

PFK1

Answer 11

regulates first committed step of glycolysis

fructose6P → fructose1,6bisP

• allosteric enzyme : regulated by many factors

regulation differs in muscle and liver…

• high glucose: lots of PFK1 activity in liver
• energy demads: lots of PFK1 activity in muscle

Question 12

allosteric regulation of PFK1

Answer 12

“high energy” molecules inhibit PFK1

• ATP
• citrate

“low energy” molecultes activate PFK1

• AMP
• fructose-2,6-bisphosphate also activates PFK1 [middleman for hormonal regulation]
  • F6P → F2,6BP via PFK2
    
    • PFK2 is upregulated by insulin via dephos, downregulated by glucagon via phos
  • F2,6BP activates PFK1 to keep glycolysis moving (F6P → F1,6BP via PFK1)

Question 13

how do we achieve the differential regulation of PFK1 in muscle and liver tissue?

Answer 13

distinct PFK2 enzymes present in muscle and in liver

• liver PFK2 follows regular hormonal reg (insulin upreg via dephos, glucagon downreg via phos)
• muscle PFK2 dependent on allosteric reg by accumulated AMP during exercise
  
  • ensures that skeletal stores of ATP, glycogen will be replenished regardless of glucose status broadcasted by hormones

Question 14

generation of NADH

Answer 14

occurs during conversion of glyceraldehyde3phosphate → 1,3bisphosphoclycerate [via glyceraldehyde3Pdehydrogenase]

Question 15

energy generation through substrate level phosphorylation

Answer 15

substrate level phos = direct transfer of P from a substrate to ADP/GDP (as opposed to ATP gen via oxphos]

1,3BPG → 3PG + ATP [via phosphoglycerate kinase]

*can also happen through intermediate conversion to 2,3BPG [R-shifter of Hb dissociation curve!]

• increased glycolysis increases 2,3BPG leading to increased O2 delivery to tissues!

Question 16

energy generation via pyruvate formation

Answer 16

phosphoenolpyruvate → pyruvate + ATP [via pyruvate kinase]

• second instance of substrate-level phosphorylation in glycolysis

Question 17

regulation of pyruvate kinase

Answer 17

allosteric regulation

• feed forward activation by fructose 1,6-bisphosphate

hormonal regulation

• insulin upreg
• glucagon downreg

Question 18

fates of pyruvate

Answer 18

cells w mitochondria and O2?

TCA cycle, ATP gen

cells without mitochondria or lacking O2?

lactate gen, regen of NAD+

• allows another round of glycolysis and gen of 2ATP
• can lead to transient lactic acidosis

Question 19

anaerobic glycolysis

Answer 19

lactate formation

• major fate of pyruvate in tissues lacking mito or w lousy vasc (lens/cornea of eye, kidney medulla, RBCs)

in exercising muscle: conversion of pyruvate → lactate

• allows glycolysis to continue by recycling NADH → NAD

in liver, heart: NADH is low

• so lactate → pyruvate, NAD → NADH

Question 20

MODY

Answer 20

maturity-onset diabetes of young

monogenic - traceable to individ mutation

• auto dominant disorder : mutations of glucokinase cause 10-65% MODY
• mild diabetes, only rarely complicated, often treated with meal planning only

Question 21

pyruvate kinase deficiency

Answer 21

• second most common cause of hemolytic anemia

RBCs lack mitochondria → are completely dep on glucose and glycolysis for egy needs

• use glucose to maintain Na/K ATPase → keeps osmotic balace which keeps cell from swelling/lysing → hemolytic anemia
• before lysis, see distorted cell membranes - characteristic spiculated appearance
• decrease in RBCs = decrease in O2 delivery = buildup of glycolytic intermeds like 2,3BPG

Question 22

causes of 2,3BPG buildup

consequence

Answer 22

2,3BPG causes O2 diss curve to shift RIGHT

• better delivery of O2 to tissues

causes

• decreased RBC count (hemolytic anemia)
• smoking (compensates somewhat for decreased O2 due to CO)
• altitude acclimatization
• COPD

Question 23

fluoride and glycolysis

Answer 23

fluoride and phosphate complex together, competitively inhibit enolase → stop glycolysis

[used to stabilize glucose in blood specimens]

Question 24

TCA cycle overview

Answer 24

occurs in mitochondria, generates energy through oxidation of acetylCoA [derived from carbs, fats, proteins] → CO2 + H2O + ATP

essential/conserved (metabolic defects are v rare)

functions

• generate free energy for ATP synthesis
  
  • fats, carbs, proteins are oxidized to CO2 → produce NADH and FADH2 for oxphos → ATP and H2O
• interconversion of fuel ⇔ metabolites
  
  • first intermediate: acetyl CoA - pdt of many catabolic pathways

Question 25

energy yield of acetyl CoA

Answer 25

fats, carbs, amino acids are metabolized to acetyl CoA each acetyl CoA yields: * 3 NADH (x3ATP) * 1 FADH2 (x2ATP) * 1 GTP (=1ATP) 1 acetyl CoA = 12 ATP

Question 26

anapleuritic reactions

Answer 26

rxns involving catabolism + anabolism ex. TCA cycle * TCA cycle generates various intermediates for cellular metabolic pathways * i.e. it's important to "refill" these intermediates so the cycle can run to completion * **anapleuritic rxns "fill in" the TCA cycle** **implication** 1. TCA intermediate levels rise and fall depending on needs of cell 2. needs of cell will influence TCA cycle via shifts in intermediate pool levels

Question 27

TCA intermediates and fates

Answer 27

**oxaloacetate:** intermediate of gluconeogenesis **citrate, oxaloacetate, acetylCoA:** membrane transport mechs **alpha ketoglutarate, oxaloacetate:** transamination rxns

Question 28

pyruvate keys: aerobic fed conditions

Answer 28

aerobic fed conditions * pyruvate → acetyl CoA [pyruvate dehydrogenase complex] * pyruvate → alanine [pyridoxal phosphate-dependent alanine aminotransferase - remember B6!]

Question 29

pyruvate keys: aerobic fasting conditions

Answer 29

aerobic fasting conditions * pyruvate → oxaloacetate [biotin-dependent pyruvate carboxylase]

Question 30

pyruvate keys: anaerobic conditions

Answer 30

anaerobic conditions * pyruvate → lactate [lactate dehydrogenase] * **in O2-deprived muscle:** pyruvate → alanine [alanine aminotransferase]

Question 31

pyruvate dehydrogenase complex

Answer 31

bridge between carbs and TCA cycle PDC converts pyruvate → acetyl CoA, which is used in TCA cycle complex of 3 enzymes: E1, E2, E3 * each is critical to complex fx * each has specific cofactors - deficiency of cofactor can produce pathology

Question 32

E1

Answer 32

pyruvate dehydrogenase component 24 chains **prosthetic**: TPP **catalyzes**: oxidative decarboxylation of pyruvate

Question 33

E2

Answer 33

dihydrolipoyl transacetylase 24 chains **prosthetic**: lipoamide **catalyzes**: transfer of acetyl group to CoA

Question 34

E3

Answer 34

dihydrolipoyl dehydrogenase [also in alphaketoglutarate dehydrogenase complex and branched chain alpha-ketoacid dehydrogenase complex] 12 chains **prosthetic**: FAD **catalyzes**: regen of oxidized form of lipoamide

Question 35

req coenzymes for PDH complex activity deficiency issues

Answer 35

**B1 (thiamine)** : thiamine pyrophosphate (TPP) **B5 (pantothenic acid)** : CoA **lipoic acid** **B2 (riboflavin)** : flavin adenine dinucleotide (FAD) **B3 (niacin)** : nicotinamide adenine dinucletide (NAD+) 4 vitamin derivatives + lipoic acid (made in sufficient amt in body) * deficiencies in any of the coenzymes affect ability to get egy from glucose → sk muscle weakness, neurological disease

Question 36

PDH complex enzyme cofactor requirements

Answer 36

_E1_ - thiamine pyrophosphate (B1 derivative) _E2_ - CoA (B5 deriv), lipoic acid * resp for step producing CoA [i.e. need for CoA] _E3_ - FAD (B2), NAD+ (B3)

Question 37

B1/thiamine deficiency

Answer 37

B1/thiamine is used by PDH, alphaketoglutarate DH, branched chain alphaketoacid DH, transketolase * deficiency affects nucleic acid synthesis, energy metabolism syndromes * beri beri * US: most common in alcoholics (poor nutrition, affects of excess alc on ability to absorb/store thiamine) * dry (muscle wasting, neuropathy) and wet (CHF + edema) * Wernicke's encephalopathy * triad of confusion, opthalmoplegia, ataxia * Korsakoff's psychosis * memory loss, confabulation, personality change

Question 38

affects of arsenite and mercury

Answer 38

* inhibit enzymes that use lipoic acid as a cofactor (including PDH complex) * CNS pathologies (ex. "mad as a hatter"!) * tx: BAL (British anti-Lewisite) - heavy metal chelator

Question 39

arsenic poisoning

Answer 39

tasteless, odorless white powder affects E2 subunit of PDH complex (and other enzymes using lipoic acid as coenzyme) _signs_ * PDC deficiency (lactic acidosis, neuro disturbances) * garlic breath, "rice-water" stools that are bloody, vomiting fun fact: Van Gogh's emerald green paint had As might have contributed to his mental episodes

Question 40

allosteric regulation of PDH complex

Answer 40

high energy moleculte **inhibit PDH** * ATP * acetyl CoA * NADH

Question 41

covalent modification : regulation of PDH complex

Answer 41

**PDH kinase** phosphorylates PDH → inactivates PDH * activated by high energy molecules **PDH phosphatase** dephosphorylates PDH → activates PDH * activated by insulin in adipose tissue * activated by Ca in muscle tissue

Question 42

dichloroacetic acid

Answer 42

synthetic analog of pyruvate * can bind to PDH kinase, inhibiting its action * prevents inhibition of PDH by keeping it in active dephos form \*might be effective treatment for lactic acidosis or MELAS, but clinical trials havent shown it

Question 43

regulation of PDH complex

Answer 43

_allosteric_ * high energy mols inhibit _covalent_ * dephosphorylation (PDH phosphatase) activates * phosphorylation (PDH kinase) inhibits * high energy mols inhibits

Question 44

PDH complex deficiency

Answer 44

* metabolic and neurologic defects * delayed devpt and reduced muscle tone * often associated with ataxia and seizures * some have congenital malformation of brain * most commonly due to mutation in X-linked E1 gene **will see accumulation of pyruvate and lactate, but normal pyruvate : lactate ratio** * glycolysis occurs, but can't do anything with the pyruvate except reduce it into lactate tx * ketogenic diet, severe restriction of protein and carb : improved mental devpt * ensures that cells use acetyl CoA from fat metabolism * if mutation affects binding of thiamine to E1, might try high dose thiamine supplementation

Question 45

metabolic and neurologic conseqs of PDH compex deficiency

Answer 45

**metabolic** : lactic acidosis (pyruvate → lactate) **neurological** : hypotonia, poor feeding, lethargy, seizures, mental retardation

Question 46

3 enzymes sharing E3...

Answer 46

1. PDH complex 2. alpha ketoglutarate DH 3. branched chain alpha ketoacid DH

Question 47

Leigh disease

Answer 47

group of disorders characterized by lactic acidosis (rare) * defects in PDH and alpha ketoglutarate complexes → cant catabolize BCAAs * incrased levels of lactate, alpha ketoglutarate, BCAAs * tissues dependent on aerobic metabolism (brain, muscle) most severely affected * impaired motor fx, neuro dorders, mental retardation

Question 48

TCA cycle overview

Answer 48

* occurs in mitochondria * all organs except ones without mito go through it * runs during fasted and fed states * aerobic * step 1: condensation of acetyl CoA + OAA → citrate * OAA regenerated at end of cycle * produces 3NADH + 1FADH2 + GTP + CO2

Question 49

irreversible reaction of TCA cycle and enzymes/regulation

Answer 49

1. acetyle CoA → citrate * **citrate synthase** 2. isocitrate → alpha ketoglutarate * **isoditrate dehydrogenase** 3. alpha ketoglutarate → succinyl CoA * **alpha ketoglutarate dehydrogenase** **regulation via allosteric activation/inhibition [NOT HORMONES]** * HIGH ENERGY mols DEACTIVATE : ATP, NADH * LOW ENERGY mols ACTIVATE: ADP, CA

Question 50

role of cellular energy in determining TCA cycle rate

Answer 50

high cellular energy → TCA cycle inhibited low cellular energy → TCA cycle activated

Question 51

allosteric regulation of TCA cycle

Answer 51

**ATP, NADH, succinyl CoA, fatty acyl derivatives** all indicate high energy * inhibit TCA cycle **ADP** indicates low energy * activates TCA cycle

Question 52

"Citrate Is Krebs' Starting Substrae For Making OAA"

Answer 52

stops on the TCA cycle * citrate * isocitrate * alphaKetoglutarate * succinyl CoA * succinate * fumarate * malate * oxaloacetate

Question 53

oxidative decarboxylation in TCA cycle

Answer 53

2 successive decarboxylations via 2 dehydrogenase rxns → release of 2 CO2 + 2 NADH * isocitrate dehydrogenase * alpha ketoglutarate dehydrogenase

Question 54

substrate level phos in TCA cycle

Answer 54

succinyl CoA → succinate [succinyl CoA thiokinase - takes off the CoA, generates a GTP) * substrate level phos producing a GTP

Question 55

3 reversible rxns to wind up TCA cycle

Answer 55

_succinate → fumarate_ [succinate dehydrogenase - FAD reduced to **FADH2 (complex II of etc)**] _fumarate → malate_ [fumarase - adds a H2O] _malate → oxaloacetate_ [malate dehydrogenase - completes recycling process, generates NADH]

Question 56

net carbon yield of TCA cycle

Answer 56

ZERO * 2 C introduced as acetyl CoA * 2 C liberated as CO2 implication: need to provide the other intermediates of the cycle in order for rxn to proceed * constant input of acetyl CoA would keep the cycle running smooth forever * TCA cycle doesn't operate in isolation... * intermediates are pulled off in other direction/rxns, need to be replaced via **anapleuritic rxns** [involve intermediates]

Question 57

energy yield from TCA cycle

Answer 57

3 NADH [NADH → NAD = 3ATP] 1 FADH2 [FADH2 → FAD = 2ATP] 1 GTP [= 1ATP] 12 ATP total

Question 58

net energy production from aerobic resp

Answer 58

1 glucose _glycolysis: 2 ATP, 2 NADH_ * each NADH from glycolysis worth either 2 or 3 ATP depending on the transporter used to shuttle it to mito 2 pyruvate _TCA cycle: 2 GTPv, 6 NADH, 2 FADH2_ **38 ATP total**