Identify the key steps in citric acid cycle

Describe how TCA is regulated

Illustrate biomedical importance of TCA

Explain energy yield from TCA.

OBJECTIVES

CITRIC ACID CYCLECITRIC ACID CYCLE (TCA CYCLE OR KREBS CYCLE)(TCA CYCLE OR KREBS CYCLE) 

PyruvatePyruvate

Acetyl-CoA Acetate CO2

Oxidizing acetyl-CoA from glucose, lipid and protein catabolism in aerobic respiration to maximize energy gain

The cycle supplies precursors for biosynthesis

CITRIC ACID CYCLECITRIC ACID CYCLE

THREE STAGES OF CELLULAR RESPIRATION

STAGE 1 Acetyl CoA production from glucose, fatty acids and amino acids

STAGE 2 Acetyl CoA oxidation =TCA Cycle = yielding reduced electron carriers

STAGE 3 Electron transport and oxidative phosphorylation oxidation of these carriers and production of ATP

MANY CATABOLIC PATHWAYS YIELDMANY CATABOLIC PATHWAYS YIELD ACETYL COA FOR THE TCA CYCLEACETYL COA FOR THE TCA CYCLE glycogenglycogen glucoseglucose lactatelactate PyruvatePyruvate fatty acidsfatty acids

amino acidsamino acids Acetyl-CoAAcetyl-CoA

TCATCA(Note: AA more than one entry point)

Acetyl CoAAcetyl CoA

HS-CoA

Space filledSpace filled

Acetyl

A high energy bond

Pyruvate (PDH) Acetyl-CoA ((PYRUVATE DEHYDROGENASE COMPLEX)PYRUVATE DEHYDROGENASE COMPLEX) 

Location = Mitochondrial matrix

CH3 CH3

C=O + NAD++ HS-CoA C=O +NADH+CO2

COO- S-CoA Pyruvate Acetyl-CoA

(A high energy compound)

STAGE 1

IRREVERSIBLE  Irreversible means acetyl-CoA cannot be converted backward to Pyruvate

Hence “fat cannot be converted tocarbohydrate”

S--S--

TPP FAD 

E1 E2 E3 N A D+

PYRUVATE DEHYDROGENASE COMPLEX

REGULATION OF PYRUVATE DEHYDROGENASE

 Irreversible reaction must be tightly controlled-- three ways1.Allosteric Inhibition

Inhibited by products: Acetyl-CoA, NADH ATP

2. Allosteric activation

AMP

Ratio ATP/AMP important

Overall Reaction in the TCA cycleOverall Reaction in the TCA cycle

ACETYL-COA + 3NAD+ + FAD + GDP + Pi+2H2O 2CO2 + 3NADH + FADH2 + GTP + 2H+

+ CoA

Both carbons oxidized One GTPThree NADHOne FADH2

1- CONDENSING ACETYL-COA WITH OXALOACETATE

 ACETYL COA

O=C-SCoA-SCoA COO- + CoASH CH3 H2O CH2 + H+

+ O=C-COO CITRATE SYNTHASE HO-C-COO-

CH2 CH2

COO- COO-

OXALOACETATE CITRATEENZYME:CITRATE SYNTHASE

2 - CITRATEISOCITRATE VIA CIS-ACONITATE

CH2-COO- -H2O CH2COO +H2O CH2-COO-

HOC-COO- C-COO H-C-COO-

CH2-COO- H-C-COO- HOC-COO-

CITRATE CIS–ACONITATE ISOCITRATE

ENZYME: ACONITASE

3- OXIDATION OF ISOCITRATE TO -KETOGLUTARATE

 First oxidation in TCA cycle

COO- COO-

CH2 NAD+ NADH CH2 + CO2

HC-COO- CH2

HOCH O=C COO- COO-

ISOCITRATE -KETOGLUTARATE

ENZYME = ISOCITRATE DEHYDROGENASE

ISOCITRATE DEHYDROGENASE

Two isoforms

One uses NAD+; other NADP+

Reduction to NADH or to NADPH

Energy is later derived from these

electron carrying molecules -- loss of first CO-- loss of first CO22

-- Note OH to =O -- Note OH to =O

4- OXIDATION OF -KETOGLUTARATE TO SUCCINYL-COA AND CO2

Second oxidation in TCA cycle

-KETOGLUTARATE SUCCINYL COA

  COO- COO- + CO2

CH2 NAD+ NADH CH2

CH2 CH2

O=C O=C COO- SCoA

+ CoA-SH ENZYME = - KETOGLUTARATE DEHYDROGENASE COMPLEX

Loss of second of two CO2

Similar to Pyruvate Acetyl-CoA

Enzyme is similar to

Pyruvate dehydrogenase complex

- KETOGLUTARATEDEHYDROGENASE COMPLEX

5- 5- SUCCINYL COA TO SUCCINATESUCCINYL COA TO SUCCINATE  

succinyl-CoA COO-

+ GDP + Pi CH2 + CoA-SH +

CH2 GTP COO-

SUCCINATE -- SUBSTRATE LEVEL PHOSPHORYLATION

-- GTP is equivalent to ATP; GTP to ATP by NUCLEOSIDE DIPHOSPHOKINASE ENZYME = SUCCINYL COA SYNTHETASE

6- OXIDATION OF SUCCINATE TO FUMARATE

FLAVIN DEPENDENT OXIDATIONThird oxidation of TCA cycle, FAD in flavoprotein reduced to FADH2

COO- COO-

CH2 + E3-FAD CH + E3-FADH2

CH2

COO- HC-COO-

SUCCINATE FUMARATE

Dehydrogenation; note double bondENZYME = SUCCINATE DEHYDROGENASE

7- 7- HYDRATIONHYDRATION

  COO- COO-

CH +H2O HOCHHC HCH COO- -H2O COO-

FUMARATE L-MALATE

ENZYME = FUMARASE

8- 8- OXIDATION OF MALATE TO OXALOACETATEOXIDATION OF MALATE TO OXALOACETATE

COO- COO-

HO HO CH NAD+ NADH C=O=O  CH2 CH2

COO- COO-

MALATE OXALOACETATE  FOURTH OXIDATION: another pair ofelectrons is made available in NADH

ENZYME = MALATE DEHYDROGENASE

SUMMARYSUMMARYFIRST HALF Introduction of two carbon atoms and their loss, yielding 2 NADH and a GTP (ATP) SECOND HALF Partial oxidation of succinate to oxaloacetate. Another NADH is produced as well as a reduced FADH2

OXALOACETATE IS REGENERATED FOR NEXT CYCLE

Overall Reaction

Acetyl-CoA+3NAD++FAD+GDP+Pi+2H2O2CO2 + 3NADH + FADH2 +GTP+2H++CoA

One high energy compound made 

Four pairs of electrons are madeavailable to the respiratory chain andoxidative phosphorylation. These areused to generate most of the ATPneeded.

What is the maximum yield of highenergy ATP in the aerobic catabolismof glucose?

GlycolysisGlycolysis::glucose 2pyruvate + 2NADH+2ATP 8 ATPs

Pyruvate Dehydrogenase:Pyruvate Dehydrogenase:2pyruvate 2acetyl CoA + 2NADH 6 ATPs

TCA cycle:TCA cycle:acetyl CoA2CO2+3NADH+FADH2+GTP 2x12ATPs OVERALL YIELD FROM GLUCOSE 38 ATPs

ENERGY RELATIONSHIPS   This represents 41% 41% conservation of

the potential energy available in

glucose as ATP

REGULATION OF CITRIC ACID CYCLEREGULATION OF CITRIC ACID CYCLE FOUR WAYS 

1- PYRUVATE DEHYDROGENASE -- Inhibited by acetyl-CoA and NADH2- CITRATE SYNTHASE -- Substrate = oxaloacetate -- limited3- ISOCITRATE DEHYDROGENASE-- Activated allosterically by ADP -- Inhibited allosterically by NADH4- 4- - KETOGLUTARATE DEHYDROGENASE- KETOGLUTARATE DEHYDROGENASE-- Inhibited allosterically by products = succinyl-CoA and NADH

Major regulator is intramitochondrial

NAD+/NADH ratio

REGULATION OF CITRIC ACID CYCLEREGULATION OF CITRIC ACID CYCLE

REPLACEMENT OF INTERMEDIATES

Intermediates are removed for

biosynthesis 

1- AMPHIBOLIC reactions (Removal of intermediates) 2- ANAPLEROTIC reactions (Replacing cyclic intermediates)

AMPHIBOLIC PATHWAYS

A- TRANSAMINASESoxaloacetate Asp removes 4C-ketoglutarate Glu removes 5Cpyruvate Ala removes 6C

B- FATTY ACID BIOSYNTHESIScitrate Acetyl CoA and oxaloacetate acetyl CoA can build fatty acids

C- HEME BIOSYNTHESISsuccinyl CoA + glycine Porphyrins

ANAPLEROTIC REACTIONS

 A-A- PYRUVATE CARBOXYLASE –Replaces oxaloacetate- most important,especially in liver and kidney

OCH3-C-COO- + CO2 + ATP O -OOC-CH2C-COO- + ADP + Pi

oxaloacetate

B- MALIC ENZYME Replaces malate-- pyruvate + CO2 +NADPHmalate + NADP+

C- FROM AMINO ACIDS

Reversals of transaminations--restores oxaloacetate or a-ketoglutarate with abundant Asp or Glu

Glutamate dehydrogenase Glu + NAD(P)+ a-ketoglutarate + NAD(P)H + NH4

+

NADH acetyl CoA NAD+ oxalo- citrate synthase MDH acetate l-malate citrate H2O fumarase aconitase H2O fumarate 2-step FADH2 succinate dehydrogenase isocitrate FAD NAD+

succinate TCATCA IDH NADHCoASH GTP succinate-CoA synthetase CO2

GDP+ Pi

succinyl CoA NADH NAD+ CO2 -ketoglutarate

  -KGDH CoASH