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PHAR150GBiochemistryDayCitricAcidCycle.pptx

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PHAR 150G

Biochemistry

TCA Cycle

Vicky Mody, PhD

[email protected]

Objectives

Identify the intracellular location of the TCA cycle and the various sources of acetyl-CoA

Describe pyruvate dehydrogenase as a multienzyme complex

List cofactors and understand mechanism of pyruvate dehydrogenase activity

List enzymes involved in TCA cycle in the proper sequence and types of reaction catalyzed

Indicate the steps that yield CO2, NADH, FADH2, and GTP

Calculate the yield of ATP from the complete oxidation of pyruvate or of acetyl-CoA

Your patient has arrived in the emergency room with alcohol-related neurological and cardiac complications. The lady that brought him in says that he has been on a binge for over a week. You would guess that some of his problems may be due to

Thiamine deficiency due to inhibition of uptake in the intestine

B12 deficiency due to excess urination

Vitamin E deficiency due to excess urination

Biotin deficiency due to excess sweating

Niacin deficiency due to the destruction of NADH by the alcohol dehydrogenase reaction

Problem

You have a patient who may have developed subclinical deficiencies of many vitamins. To be sure that the pyruvate dehydrogenase and the pyruvate carboxylase reactions would have an adequate amount of cofactors, you would prescribe all of the following EXCEPT

Vitamin D3

Pantothenate

Riboflavin

Thiamine

Problem

Disorder	Cause	Explanation
Anorexia Nervosa	Genetic/Enviornmental	Patients who have been malnourished for some time may exhibit subclinical deficiencies in many vitamins, including riboflavin and niacin, factors required for energy generation.
Beri Beri	Thiamine Deficiency	Can be due to high intake of Alcohol or genetic

Diseases discussed in this Class

Must Know

Disorder	Cause	Explanation
Conjestive Heart Failure Linked to Alcoholism	Genetic/Enviornmental	Thiamine deficiency brought about by chronic alcohol ingestion leads to inefficient energy production by the heart and failure to adequately pump blood throughout the body. The vitamin B1 deficiency reduces the activity of pyruvate dehydrogenase and the TCA cycle, severely restricting ATP generation.
Arsenic Poisoning	Genetic/Enviornmental	Arsenite inhibits enzymes and cofactors with free adjacent sulfhydral groups (lipoic acid is a target of arsenite), whereas arsenate acts as a phosphate analog and inhibits substrate-level phosphorylation reactions.

Diseases discussed in this Class

Must Know

Disorder	Cause	Explanation
Leigh disease (subacute necrotizing encephalopathy)	Genetic	Deficiencies of the pyruvate dehydrogenase complex (PDC) as well as of pyruvate carboxylase are inherited disorders leading to lactic acidemia. In its most severe form, PDC deficiency presents with overwhelming lactic acidosis at birth, with death in the neonatal period. Even in less severe forms, neurologic symptoms arise due to the brain’s dependence on glucose metabolism for energy. The most common PDC deficiency is X-linked, in the a-subunit of the pyruvate decarboxylase (E1) subunit. Pyruvate carboxylase deficiency also leads to mental retardation.

Diseases discussed in this Class

Must Know

Stage I All the fuel molecules (glucose, amino acids, fatty acids) are oxidized to acetyl-CoA.

Stage II The acetyl-CoA is oxidized into CO2, electrons are collected by NAD and FAD via the citric acid cycle.

Stage III Transfer of electrons to O2 through the electron transport system to yield ATP from oxidative phosphorylation.

Three Stages of Respiration

Make ATP.

Make high energy electron carriers that can transfer electrons to O2 in the electron transport chain for use in oxidative phosphorylation.

NADH

FADH2

Overall Goal

Read up about substrate level phosphorylation and oxidative phosphorylation

ATP is the energy currency of the cell and much like the 1 dollar bill, it has a fixed value and once it is used up we need to replenish it. A single heartbeat uses about 2% of all the ATP content of the heart.. All the heart store of ATP will be used up in 1 minute if not replenished

No net gain in Carbon during the TCA, 8electrons.

If cells don’t have the ability ot go through the cycle, then thre are toast because this cycle produces a lot of energy about 2/3 of the energy from glucose. If we inhibit the cycle , the cell die.

Intermediates from TCA are intermediates in other cycles- it is an anaplerotic. Eights steps in this cycle, shorter than glycoslysis that has 10

Glycolysis in the cytosol

TCA takes place in the matrix

of Mitochondrion

Outer membrane is permeable

Space between membranes called intermembrane space

Inner membrane is impermeable to anions and cations

Geography

Why is this important?

Importance of Vitamins

4 Vitamins are key players in the TCA cycle

Thiamin (Vitamin B1)

As coenzyme (Thiamine pyrophosphate, TPP) in pyruvate dehydrogenase complex (PDC) and α-ketoglutarate dehydrogenase (KDH) catalyzed reactions

Riboflavin (Vitamin B2)

As flavin adenine dinucleotide (FAD)

Niacin (Vitamin B3)

As nicotinamide adenine dinucleotide (NAD+)

Pantothenic acid (Vitamin B5)

As part of coenzyme A

Must Know

Patients who have been malnourished for some time may exhibit subclinical deficiencies in many vitamins, including riboflavin and niacin, factors required for energy generation.

Anorexia Nervosa and Vitamin Deficiency

Must Know

Anorexia Nervosa and Vitamin Deficiency

Must Know

Link Between Glycolysis and TCA

Pyruvate Dehydrogenase Complex (PDC)

Links glycolysis to TCA cycle

Catalyzes the irreversible (ΔG = -33.4kJmol) conversion (decarboxylation) of pyruvate to AcetylCoA

Arsenic and alcohol interferes with PDH and causes poisoning

Must Know Very Important In Cancer too

Under anaerobic conditions pyruvate is converted to lactate or ethanol depending on the organism

Large, integrated complex and it requires 5 coenzymes (NAD+ and CoA and three that are enzyme associated)

Made up of 3 distinct enzymes

Pyruvate decarboxylase (E1)

Requires Thiamine pyrophosphase as cofactor (Vitamin B1)

Dihydrolipoyl transacetylase (E2)

Requires Lipoic acid as cofactor

Dihydrolipoyl dehydrogenase (E3)

Requires FAD (Vitamin B2) as cofactor

Pyruvate Dehydrogenase Complex

(NAD+ from Vitamin B3)

Must Know Very Important In Cancer too

Leigh Syndrome

Deficiencies of the pyruvate dehydrogenase complex (PDC) as well as of pyruvate carboxylase.

This leads to accumulation of lactic acid as pyruvate cannot be metabolized by TCA

Must Know

Leigh Syndrome

In its most severe form, PDC deficiency presents with overwhelming lactic acidosis at birth, with death in the neonatal period.

Even in less severe forms, neurologic symptoms arise due to the brain’s dependence on glucose metabolism for energy.

The most common PDC deficiency is X-linked, in the a-subunit of the pyruvate decarboxylase (E1) subunit.

Pyruvate carboxylase deficiency also leads to mental retardation.

Must Know

Leigh Syndrome

Must Know

Acetyl CoA in TCA and its various Sources

AcetylCoA is a Central Molecule in Energy Production

Must Know Very Important

Regulation of the Pyruvate Dehydrogenase Complex

Controlled primarily by phosphorylation

Pyruvate dehydrogenase kinase

Inactivates PDC

Pyruvate dehydrogenase phosphatase

Activates PDC

Must Know and understand how this cycle works

Regulation of the Pyruvate Dehydrogenase Complex

Pyruvate dehydrogenase kinase

Inhibited by ADP, NAD+ and pyruvate

Activated by Acetyl-coA and NADH

Pyruvate dehydrogenase phosphatase

Activated by Ca2+

Important during muscle contraction

Elevated intracellular Ca2+

Phosphatase activated activated PDC more acetyl-CoA available for the TCA cycle increased energy production.

Must Know and understand how this cycle works

Pyruvate Dehydrogenase Kinase in Cancer Patients

Must Know Very Important

Citric Acid Cycle

Main function: to oxidize Acetyl CoA to CO2 while conserving energy in the form of NADH, FAD(2H) and GTP

Harvesting high energy electrons from carbon fuels

TCA cycle accounts for 2/3rds of total ATP production

Only under aerobic conditions

Important in synthesis reactions

Occurs in mitochondrial matrix

Citric Acid Cycle Overview

Step 1 Formation of Citrate

Must Know the cycle

No structures

Formation of Citrate

Condensation reaction

Irreversible

Citrate Synthase

Step 2

Citrate to Isocitrate

Hydroxyl moved and changed from tertiary (alcohol) to secondary.

Isomerization involving dehydration (elimination) followed by hydration (addition)

Aconitase

Tertiary cannot be oxidized hence the need for rearrangement to give secondary which can be oxidized

Inhibition of Aconitase

Fluoracetate Inhibits Aconitase

Fluoroacetate is a natural form of toxic compound sodium fluoroacetate,

Also known as the notorious rodent poison 'Compound 1080'.

When ingested, fluoroacetate is transformed in cells to fluoroacetylCoA and ultimately fluorocitrate – a potent Aconitase inhibitor.

Fluorocitrate blocks the TCA Cycle completely!

Must Know very Important

Step 3

Isocitrate to a-Ketoglutarate

Isocitrate to α-Ketoglutarate

Oxidative decarboxylation

Rate limiting step and Irreversible

Isocitrate dehydrogenase

Isocitrate+ NAD+

Isocitrate dehydrogenase

α-Ketoglutarate + CO2 + NADH

Step 4

a-Ketoglutarate to Succinyl CoA

α-Ketoglutarate to SuccinylCoA

A-ketoglutarate and dehydrogenase complex uses same enzymes and coenzymes as PDC

Oxidative decarboxylation

Irreversible

α-Ketoglutarate dehydrogenase

complex

Must Know the similarity between PDC and KDC

Step 5

Succinyl CoA to Succinate

SuccinylcoA to Succinate

Substrate Level Phosphorylation - direct donation of Pi to ADP or GDP to form ATP to GTP.

SuccinylCoA synthetase (Succinate thiokinase)

Hydrolysis of the high energy thioester bond provides the energy that drives the addition of phosphate to GDP to give GTP

SuccinylCoA Synthetase

Step 6

Succinate to Fumarate

Oxidation

FAD and FADH2 remain enzyme bound at all times

Arsenic and Malonate inhibits SDH

Succinate Dehydrogenase

Succinate + FAD

Succinate dehydrogenase

Fumarate + FADH2

NAD+ is used to oxidize oxygen-containing groups (Aldehydes and alcohols)

FAD is used to oxidize C-C bonds

Step 7

Fumarate to Malate

Hydration

Fumarase

Step 7

Malate to Oxaloacetate

Oxidation

Oxidation of secondary alcohol to ketone

Regenerates oxaloacetate for another round

Malate + NAD+

Malate

dehydrogenase

Oxaloacetate + NADH + H+

Malate Dehydrogenase

Thermodynamics of the TCA Cycle

TCA Cycle is energetically favorable and largely driven by the three irreversible reactions

Oxidative process

3 NADH

FADH2

GTP

1 glucose gives 2- pyruvate which gives 2- acetylCoA

X 2

6 NADH

2 FADH2

2 GTP

All ultimately turned into ATP via oxidative phosphorylation

Net from TCA Cycle (Acetyl-CoA)

In Mitochondrion

From Pyruvate dehydrogenase Reaction

2 NADH

From TCA Cycle

6 NADH

2 FADH2

2 GTP

Hence : 8 NADH, 2 FADH2, 2 GTP from Mitochondria

Total Energy Starting from Pyruvate

In mitochondrion:

Each NADH makes 2.5 ATP

Each FADH2 makes 1.5 ATP

GTP makes ATP

So…

From both PDC and Citric acid Cycle;

8 NADH X 2.5 ATP/NADH = 20 ATP

2 FADH2 X 1.5 ATP/FADH2= 3 ATP

2 GTP X 1 ATP / GTP = 2 ATP

TOTAL in mitochondrion 25 ATP

Total Energy from Pyruvate

Must Know

The enzymes have greater access to products of the previous reaction.

Molecules need special transporters

Pyruvate moves via the mitochondrial pyruvate carrier (mpc)

NAD+, NADH, CoASH and acetylcoA have no transport proteins- cannot cross the membrane

Allows for tight control of the TCA cycle (to be discussed later)

Q: How is NADH from glycolysis is converted to NAD+ if it cannot cross into mitochondria.

Geography (Cont.)

NADH from Glycolysis is transferred via Malate Aspartate Cycle

Anaplerotic Reactions (“filling up”)

Conversion of pyruvate to oxaloacetate by Pyruvate carboxylase

PropionylCoA converted to SuccinylcoA (3)

Amino acid degradation (2,4 and 5)

PC is activated by acetylcoA

Diseases Due to Disruption of TCA Cycle

Beriberi : deficiency of thiamin (B1)

Genetic or due to chronic alcohol consumption

Alcohol

Impairs thiamine absorption from the G.I

Impairs utilization by cells

Reduction in activity of PDC and ketoglutarate dehydrogenase (Why?)

Severe reduction in ability to produce ATP

Neurological deficits

Wernicke–Korsakoff syndrome

Muscle weakness and paralysis

Inability to pump blood effectively to the body

Congestive heart failure

Must Know

Each heart beat uses about 2% of the total ATP in the heart

Thiamine deficiency due to inhibition of uptake in the intestine

B12 deficiency due to excess urination

Vitamin E deficiency due to excess urination

Biotin deficiency due to excess sweating

Niacin deficiency due to the destruction of NADH by the alcohol dehydrogenase reaction

Problem

Your patient is an alcoholic a buildup of a-ketoacids and symptoms of wet beriberi. All of the following might be part of a scenario that would explain why peripheral vessels dilate and cardiac muscles loose their contractility EXCEPT

Most ATP is produced by oxidative phosphorylation

NADH and FADH2 are produced by the TCA cycle

The TCA cycle needs thiamine to function

A shortage of thiamine results in the inability to oxidize NADH to NAD+ needed for TCA cycle

Problems

Vitamin D3

Pantothenate

Riboflavin

Thiamine

Problem

Summary

Identify the intracellular location of the TCA cycle and the various sources of acetyl-CoA

Describe pyruvate dehydrogenase as a multienzyme complex

List cofactors and understand mechanism of pyruvate dehydrogenase activity

List enzymes involved in TCA cycle in the proper sequence and types of reaction catalyzed

Indicate the steps that yield CO2, NADH, FADH2, and GTP

Calculate the yield of ATP from the complete oxidation of pyruvate or of acetyl-CoA