Exam 02 Combined Lectures

What does PDHC do?

Converts pyruvate → acetyl-CoA for TCA entry

What are pyruvate’s fates under aerobic vs. anaerobic conditions?

Anaerobic: lactate (animals), ethanol (microbes) and aerobic: acetyl-CoA (PDHC)

Why is TCA cycle important? 

Extracts energy from acetyl groups; produces NADH/FADH2 to power ETC (32-38 ATP) 

Total electron yield from glucose oxidation? 

24 electrons → via NADH & FADH2 

How does pyruvate enter mitochondria?

Pyruvate/H+ symporter (inner membrane); porins (outer membrane)

Why co-transport H+?

Supports proton motive force for ATP synthesis

Overall PDHC reaction? 

Pyruvate + CoA + NAD+ → Acetyl-CoA + CO2 + NADH 

Enzymes & cofactors in PDHC?

E1: Pyruvate Dehydrogenase — TPP. E2: Dihydrolipoyl Transacetylase — Lipoamide, CoA. E3: Dihydrolipoyl Dehydrogenase — FAD, NAD+

Role of E1?

Decarboxylates pyruvate → hydroxyethyl-TPP intermediate

What does TPP do? 

Stabilizes carbanion intermediate; from Vitamin B1 (deficiency → BeriBeri) 

Role of E2?

Transfers hydroxyethyl from TPP to CoA → makes acetyl-CoA

What’s special about Lipoamide?

Swinging arm between E1/E2/E3; oxidized form receives intermediate, reduced form releases

Role of E3? 

Reoxidizes lipoamide using FAD → FADH2 → NADH 

Final electron acceptor in PDHC?

NAD+ (becomes NADH)

Structure of PDHC?

E2 core (60 subunits), surrounded by E1 and E3; multienzyme complex

Advantages of multienzyme complexes? 

Coordinate catalysis, channel intermediates, prevent side reactions, efficient conversion 

PDHC regulation—Allosteric?

Inhibited by NADH, acetyl-CoA, ATP, fatty acids, activated by AMP, Ca2+

PDHC regulation—Covalent?

PDH kinase phosphorylates → inhibits (high energy state). PDH phosphatase dephosphorylates → activates (low energy state)

Vitamin sources for PDHC cofactors? 

TPP (Vit B1), Lipoate (FA precursor), CoA (B5), FAD (B2), NAD+ (B3) 

PDHC is the…

bridge between glycolysis and TCA.

Produces 1 NADH + 1 CO2 per…

pyruvate

PDHC is regulated tightly by…

energy state (ATP/NADH levels) and phosphorylation 

What does the TCA cycle do? 

Oxidizes Acetyl-CoA → CO2, generates 3 NADH, 1 FADH2, and 1 GTP per cycle 

Who discovered the TCA cycle?

Hans Krebs; also characterized the Urea and Glyoxylate cycles

How does pyruvate enter the mitochondria?

Pyruvate/H+ symporter using proton motive force (-220mV)

Why is membrane potential important?

Drives metabolite transport, maintain cell viability

Inputs and outputs per cycle? 

Aceyl-CoA → 2 CO2, 3 NADH, 1 FADH 2, 1 GTP 

2 main goals of TCA cycle?

Energy production (ETC/ATP) and biosynthetic precursors

Reaction 01

Citrate Synthase

What happens in R1? 

Acetyl-CoA (2C) + OAA (4C) → Citrate (6C); thioester hydrolysis drives reaction; irreversible 

Enzyme & regulation? 

Citrate Synthase; highly exergonic and regulated 

Purpose of R2?

Isomerize citrate → isocitrate (secondary alcohol, better substrate for oxidation)

Cofactor used in reaction 02?

Fe—S cluster for stereospecific dehydration/rehydration

Reaction 03

Isocitrate Dehydrogenase 

Reaction & products of reaction 03?

Isocitrate → alpha-ketoglutarate + CO2 + NADH (1st oxidative decarboxylation)

Regulation of reaction 03?

Inhibited by NADH, ATP; activated by AMP

Reaction 04 

alpha-Ketoglutarate Dehydrogenase 

Products of reaction 04? 

alpha-KG → Succinyl-CoA + CO2 + NADH (2nd oxidative decarboxylation) 

Cofactors of reaction 04?

TPP, Lipoamide, CoA, FAD, NAD+ (like PDHC)

Regulation of reaction 04?

Inhibited by NADH, succinyl-CoA; activated by Ca2+

Reaction 05

Succinyl-CoA Synthetase

What happens in reaction 05?

Succinyl-CoA → Succinate + GTP (substrate-level phosphorylation)

Why is ΔG near 0?

Thioester hydrolysis energy is coupled to GTP synthesis

Reaction 06 

Succinate Dehydrogenase 

Products and cofactor of reaction 06?

Succinate → Fumarate + FADH 2

Why FAD instead of NAD+ for reaction 06?

Reduction potential make NAD+ transfer unfavorable (+ 66.8 kJ/mol); FAD is more favorable (+ 13.7 kJ/mol)

Enzyme special feature in reaction 06? 

Also Complex II of ETC 

Reaction 07

Fumarase

What happens in reaction 07?

Fumarate + H2O → Malate (hydration; trans stereochemistry)

Reaction & significance? 

Malate → OAA + NADH; unfavorable but pulled forward by citrate synthase consumption of OAA 

Net products per Acetyl-CoA?

3NADH, 1FADH2, 1 GTP, 2 CO2

Main exergonic steps (regulation points)? 

Citrate synthase, IDH, alpha-KGDH 

Which metabolites are used for biosynthsis?

alpha-KG (→ amino acids), OAA (→ aspartate), citrate (→ FA synthesis), succinyl-CoA (→ heme)

What are anaplerotic reactions?

Reactions that refill TCA intermediates (i.e., pyruvate carboxylase → OAA)

Why shuttles? 

Move NADH equivalents from cytosol to mitochondria 

Types of shuttles?

Malate-Aspartate Shuttle (NADH retained), Glycerophosphate Shuttle (NADH → FADH2)

How does glyoxylate cycle differ from TCA?

Bypasses decarboxylation steps; uses isocitrate lyase + malate synthase to converse carbons

— enzymatic steps per cycle. 

8 

Major control points:

Citrate Synthase, IDH, alpha-KGDH

TCA yields…

3 NADH, 1 FADH2, 1 GTP per Acetyl-CoA

TCA is amphibolic. What does this mean? 

It is used for energy AND biosynthesis 

What activate PDHC allosterically?

Low energy signals: ADP, NAD+, CoA, Ca2+

What inhibits PDHC allosterically?

High energy signals: ATP, NADH, Acetyl-CoA, fatty acids

How is PDHC inactivated covalently? 

Phosphorylation of E1 by PDH kinase → block decarboxylation 

How is PDHC activated covalently? 

Dephosphorylation by PDH phosphatase

PDH kinase regulation?

Activated by high energy (ATP/NADH); inhibited by low energy (ADP/ NAD+)

PDH phosphatase regulation?

Activated by insulin and Ca2+

Inhibitors 

ATP, NADH, Acetyl-CoA, fatty acids

Activators

ADP, NAD+, CoA, pyruvate, insulin Ca2+

How many enzymes in TCA are regulated?

3: Citrate Synthase, Isocitrate Dehydrogenase (IDH), alpha-Ketoglutarate Dehydrogenase (alpha-KGDH)

Primary allosteric inhibitor of TCA? 

NADH (feedback inhibition) 

Other feedback regulators?

Citrate, Succinyl-CoA, ATP

Activators of Citrate Synthase?

Low energy signals (ADP, NAD+)

Inhibitors of Citrate Synthase? 

High energy signals (ATP, NADH), citrate, succinyl-CoA 

Activators of IDH?

ADP, NAD+, CA2+

Inhibitors of IDH?

ATP, NADH

Activators of alpha-KGDH? 

Ca2+ 

Inhibitors of alpha-KGDH?

ATP, NADH, Succinyl-CoA

What factors influence TCA cycle flux?

ETC activity, NAD+ regeneration, oxygen availability, ATP levels

PDHC regulated by…

energy status + covalent phosphorylation 

TCA regulated mainly at…

Citrate Synthase, IDH, and alpha-KGDH

NADH =

main feedback inhibitor

— is a strong activator, especially during muscle contraction 

Ca2+ 

What affects TCA turnover?

ETC flux

Main function of ETC?

Extract electrons from NADH & FADH2 → transfer them to O2 → generates proton gradient → power ATP synthesis

Where is the ETC located?

Inner mitochondrial membrane; proton pumped from matrix to intermembrane space 

Why is ETC a multistep process?

Electron carriers increase in affinity stepwise → allows controlled transfer proton pumping, and less ROS

ΔG° of NADH → O₂ transfer?

-219 kJ/mol (NADH); -164 kJ/mol (FADH2) — favorable

Direction of electron flow?

NADH/FADH2 → FMN/Fe-S → CoQ → Cyt C → O2

Electron source and flow in complex I? 

NADH donates 2e- → FMN → Fe-S clusters → CoQ → QH2

How many protons are pumped at complex I?

4 H+

Key prosthetic groups in complex I?

FMN (2e-) and Fe-S cluster (1e- tansfers)

Electron source for Complex II? 

FADH2 from succinate dehydrogenase reaction 

Protons pumped at Complex II?

0 H+ (why FADH2 yields less ATP)

Electron source for complex III?

QH2 generated from Complex I and II

Mobile electron carrier produced? 

Cytochrome C (1e- carrier) 

What is the Q cycle?

2-cycle mechanism splitting QH2 electrons: 1 goes to Cyt, 1 regenerates QH2; pumps total of 4 H+

Final electron acceptor (in complex IV)?

O2 → reduced to H2O

How many electrons needed to reduce O2? 

4 electrons from 4 Cyt C

Protons pumped at Complex IV?

2H+