EXAM 2: Part 3 - Tricarboxylic Acid Cycle

Location?

Inside mitochondria

TCA

shared metabolic pathway of all fuels
Oxidatively strips e- from Acetyl CoA → produces majority of reduced coenzymes (carrying e-) that are used for ATP generation ini e-transport chain
Does not need O2, but needs oxidative metabolism

TCA functions

E production
Biosynthesis (3NADH, 1 FADH2)

Prep for Cycle PART 1

When O2 present, pyruvate (3Carbon) → Acetyl CoA (2 carbon)
TCA accepts 2C acetyl units in form of Acetyl CoA and binds to a 4C acceptor
2C units are oxidized to CO2 and High E e-s are captured
4C acceptor regenerated

What happens when pyruvate is transported to mitochondria matrix?

Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase → Acetyl CoA

The conversion of pyruvate → Acetyl CoA is…

Irreversible and is the link btwn glycolysis and TCA

Pyruvate dehydrogenase complex products:

CO2, plus captures high transfer E e- in NADH form

TCA’s required enzymes

Pyruvate dehydrogenase, Dihydrolipoyl transacetylase, Dihydrolipoyl dehydrogenase
5 required coenzymes

Catalytic coenz

not permanently altered by rxns

Stoichiometric coenz

act as substrates

Steps of TCA

1. Decarboxylation

2. Oxidation

3. Transfer of resulting acetyl group CoA

Pyruvate dehydrogenase complex’s domains

1. Pyruvate dehydrogenase

2. Transacetyl core

3. Dihydrolipoyl dehydrogenase

Note, if 1 is inhibited, the complex stops working

Pyruvate dehydrogenase complex

3 kinds of enzymes and long flexible lipoamide arm allow coordinated catalysis of complex rxns
Proximity increases rxn rates and decreases side rxns

PDH regulation

Regulated by 2 mechs:
Regulated by allosteric interactions and covalent mechanisms
High [products] inhibit rxn → NADH, Acetyl CoA, ATP
Key means = covalent modification → PHOSPHORLATION

Inhibition of PDH has what effect on glucose

Sparing of glucose

PDH deactivation reversal caused by

PDH Phosphatase

PDH activated by

pyruvate and ADP

At rest, E charge is ___

High

At a high E charge, the PDH is …

Off

Harvesting e-s from cycle (PART 2)

Acetyl CoA is fuel
Series of ox red rxns
Oxidation generates high transfer potential/high E e-s that power synthesis of ATP

Subparts of Harvesting stage

Oxidation of C atoms → CO2
Regeneration of oxaloacetate

Oxaloacetate

4C component
→ 6C tricarboxylic acid = CITRATE

Citrate

Releases 2 CO2, yields high E e-s
Now, 1 4C compound remains —oxidation→ regenerates oxalacetate

Oxidative decarboxylation

2Cs come in with acetyl unit, 2 leave as CO2
Regens oxaloacetate

TCA and citrate

TCA captures high E e-s from citrate and forms NADH and FADH2
E- carriers yield 9 molec of ATP

p+ gradient →

generates ATP from ADP

STAGE 1

Oxidation of C → CO2

Citrate synthase

condensation of oxaloacetate w/acetyl group from Acetyl CoA
intermediate = citryl CoA = Energy rich thioester

Citrate synthase preventing waste

oxaloacetate binds first and induces major structural changes
Citryl CoA induces more change
This is an example of the Induced Fit Model

Isocitrate

Isomerizes citrate, allows oxidation
It occurs bc citrate is not optimally suited for this (must be isomerized)
1st of 4 oxidation-reduction rxns

Isocitrate dehydrogenase

catalyzes isocitrate and allosteric regulators
Also produces NADH

When isocitrate binds to enzyme…

Loses CO2 to form a-ketoglutrate
first generation of a high-transfer-potential electron carrier, NADH

Soccinyl CoA is formed by oxidative decarbox of an a-ketoglutarate

2nd ox reduction
Removes a CO2 from a-ketoglutarate → forms succinyl CoA
Catalyzed by a-ketoglutarate dehydrogenase complex
Another NADH forms
At this point, 2Cs have entered TCA and both oxidized to CO2. e-s both captured in 2 NADH

a-ketoglutarate dehydrogenase complex

catalyzes oxidative decarbox of an a-ketoglutarate → Soccinyl CoA

STAGE 2

Regeneration of oxaloacetate
ONLY step in TCA that directly yields high phosphoryl-transfer potential

2 forms of enzyzme

ADP, GDP

ADP

Skeletal and heart muscle

GDP

tissues that perform many anabolic rxns like liver

Succinate is oxidized to regenerate …

Oxaloacetate
3 steps

More E extracted in form of …

FADH2 and NADH

TCA net rxn/products

3 NADH and FADH2
2C atoms enter and 2 leave as CO2
4 pairs of H atoms leave in 4 oxidation rxn plus p+ gradient
1 ATP/GTP
2 H2O consumed

Key catabolic function is production of high E e-s in form of NADH and FADH2
Strictly aerobic

Enzymes in TCA and connecting channels

Enzs are physically assoc w/one another
One active site connected to the next via SUBSTRATE CHANNELING

Primary Allosteric enz of control

Isocitrate dehydrogenase
a-ketoglutarate dehydrogenase

Stimulates / Inhibits Isocitrate dehydrogenase

Stim ADP / Inhib is NADH + ADP

Function + inhibition of a-ketoglutarate dehydrogenase

Catalyzes rate limiting step in TCA
Inhibited by succinyl CoA, NADH, ATP

How oxaloacetate is replenished

formed by carboxylation of pyruvate by Pyruvate carboxylase
Anaplerotic rxn → leadds to net synthesis/replenishment of pathways components

If E charge is high…

Oxaloacetate → glucose

If low E charge…

oxaloacetate replenishes TCA

T/F TCA cycle can be replenished by generation of ANY intermediate

True

Removal of N

Glutamate → a-ketoglutarate
Asparate → oxaloacetate

Glutamine can be converted into →

glutamate → α-ketoglutarate

SUMMARY

PDH — pyruvate → Acetyl CoA
PDH complex regulated by 2 mechs— allosteric inhib by products and covalent modification (phosphorylation)
Cycle has 2 stages:
— I - oxidized 2 Cs to gather E rich e-s
— II - regenerates oxaloacetate and harvests E rich e-s
Cycle is highly regulated