Krebs Cycle

Dr. Leena S Barhate

Acknowledgement

• www.worldofteaching.com

• www2.fiu.edu/~bch3033/Handouts/Lh6Ch16TCA.ppt

• www.uh.edu/sibs/faculty/glegge/lecture_23a.ppt

• cronus.uwindsor.ca/units/biochem/web/biochemi.nsf/.../Citric%20acid%20cycle.ppt

Discovered CAC in Pigeon Flight Muscle

The Citric acid cycle

It is called the Krebs cycle or the tricarboxylic and is the “hub” of the metabolic system. It accounts for the majority of carbohydrate, fatty acid and amino acid oxidation. It also accounts for a majority of the generation of these compounds and others as well.

Amphibolic - acts both catabolically and anabolically

3NAD+ + FAD + GDP + Pi + acetyl-CoA

3NADH + FADH + GTP + CoA + 2CO2

• 1937: Krebs: Enzymatic conversion of Pyruvate + Oxaloacetate to citrate and CO2

• Discovered the cycle of these reactions and found it to be a major pathway for pyruvateoxidation in muscle.

In Mitochondria

In Cytosol

Reaction of pyruvate dehydrogenase complex (PDC)

Thiamine pyrophosphate

Flavin adenine dinucleotidePyruvate dehydrogenaseDihydrolipoyl transacetylaseDihydrolipoyl dehydrogenase

Acetyl-lipoamide

Pyruvate dehydrogenase Complex (PDC)

It is a multi-enzyme complex containing three enzymes associated together non-covalently:

E-1 : Pyruvate dehydrogenase , uses Thiamine pyrophosphate as cofactor bound to E1

E-2 : Dihydrolipoyl transacetylase, Lipoic acid bound, CoA as substrate

E-3 : Dihydrolipoyl Dehydrogenase FAD bound, NAD+ as substrate

Advantages of multienzyme complex:

1. Higher rate of reaction: Because product of one enzyme acts as a substrate of other, and is available for the active site of next enzyme without much diffusion.

2. Minimum side reaction.3. Coordinated control.

The Krebs Cycle

• Occurs in the matrix of the mitochondrion

• Aerobic phase (requires oxygen)

• 2-carbon acetyl CoA joins with a 4-carbon compound to form a 6- carbon compound called Citric acid

• Citric acid (6C) is gradually converted back to the 4-carbon compound-ready to start the cycle once more

• The carbons removed are released as CO2

-enzymes controlling this process called decarboxylases

• The hydrogens, which are removed, join with NAD to form NADH2 -enzymes controlling the release of hydrogen are called dehydrogenases

Citric Acid Cycle

Summary of Krebs CycleHow many energy-producing molecules do we have per 1 glucose molecule?Intermediate step: Pyruvate oxidation1 NADH x2= 2 NADH

Krebs Cycle: 3 NADHx2= 6 NADH1 ATPx2= 2 ATP1 FADH2x2= 2 FADH2

+ 2 ATP, 2 NADH from glycolysis

Total: 4 ATP, 10 NADH, 2 FADH2 --> forms 38 ATP in the electron transport chain

ATP

ATP

ATP

ATP

Gluconeogenesis;

gluco neo genesis

sugar (re)new make/create

glycolysis

glucose

pyruvateLactateAmino acidglycerol

gluconeogenesis

The source of pyruvate and oxaloacetate for gluconeogenesis during fasting or carbohydrate starvation is mainly amino acid catabolism.

Some amino acids are catabolized to pyruvate, oxaloacetate, or precursors of these.

Muscle proteins may break down to supply amino acids. These are transported to liver where they are deaminated and converted to gluconeogenesis inputs.

Glycerol, derived from hydrolysis of triacylglycerols in fat cells, is also a significant input to gluconeogenesis.

27

Dietary & muscle

proteins

Amino acids

Triglycerols

glycerol

Fatty acids

Noncarbohydrate precursors of glucose

28

Main sites of gluconeogenesis:

• Major site: Liver.• Minor site: Kidney.• Very little:

– Brain.– Muscle (skeletal and heart).

In liver and kidney it helps to maintain the glucose level in the blood so that brain and muscle can extract sufficient glucose from it to meet their metabolic demands.

29

Gluconeogenesis Versus Glycolysis:

• 7 steps are shared between glycolysis and gluconeogenesis.

• 3 essentially irreversible steps shift the equilibrium far on the side of glycolysis.

• Most of the decrease in free energy (consuming energy) in glycolysis takes place during these 3 steps.

• These steps must be bypassed in Gluconeogenesis.

• Two of the bypass reactions involve simple hydrolysis reactions.

30

31

Hexokinase or Glucokinase (Glycolysis) catalyzes:

glucose + ATP glucose-6-phosphate + ADP

Glucose-6-Phosphatase (Gluconeogenesis) catalyzes:

glucose-6-phosphate + H2O glucose + Pi

HO

O H

H

O HH

O H

C H 2 O H

H

O H

HHO

O H

H

O HH

O H

C H 2 O P O 32

H

O H

H

H 2 O1

6

5

4

3 2

+ P i

g lu c o s e -6 -p h o s p h a te g lu c o s e

G lu c o s e -6 -p h o s p h a ta s e

Glucose-6-phosphatase enzyme is embedded in the endoplasmic reticulum (ER) membrane in liver cells.

The catalytic site is found to be exposed to the ER lumen. Another subunit may function as a translocase, providing access of substrate to the active site.

HO

O H

H

O HH

O H

C H 2 O H

H

O H

HHO

O H

H

O HH

O H

C H 2 O P O 32

H

O H

H

H 2 O1

6

5

4

3 2

+ P i

g lu c o s e -6 -p h o s p h a te g lu c o s e

G lu c o s e -6 -p h o s p h a ta s e

Phosphofructokinase (Glycolysis) catalyzes:

fructose-6-P + ATP fructose-1,6-bisP + ADP

Fructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes:

fructose-1,6-bisP + H2O fructose-6-P + Pi

f ru c to se -6 -p h o s p h a te f ru c to se -1 ,6 -b is p h o s p h a te

P h o s p h o fru c to k in a s e

C H 2 O P O 32

O H

C H 2 O H

H

O H H

H H O

O

6

5

4 3

2

1 C H 2 O P O 32

O H

C H 2 O P O 32

H

O H H

H H O

O

6

5

4 3

2

1

A T P A D P

P i H 2 O

F ru c to s e -1 ,6 -b io s p h o s p h a ta s e

Bypass of Pyruvate Kinase:

Pyruvate Kinase (last step of Glycolysis) catalyzes:

phosphoenolpyruvate + ADP pyruvate + ATP

For bypass of the Pyruvate Kinase reaction, cleavage of 2 ~P bonds is required.

DG for cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP).

PEP has a higher negative DG of phosphate hydrolysis than ATP.

Bypass of Pyruvate Kinase (2 enzymes):

Pyruvate Carboxylase (Gluconeogenesis) catalyzes:

pyruvate + HCO3 + ATP oxaloacetate + ADP + Pi

PEP Carboxykinase (Gluconeogenesis) catalyzes:

oxaloacetate + GTP PEP + GDP + CO2

C

C

CH 2

O O

O PO 32

C

C

CH 3

O O

O

A T P A D P + P i C

CH 2

C

C

O

O O

O

O

HC O 3

G T P G D P

CO 2

p y ru v a te o x a lo a c e ta te P E P

P y ru v a te C a rb o x y la s e P E P C a rb o x y k in a s e

Contributing to spontaneity of the 2-step process:

Free energy of one ~P bond of ATP is conserved in the carboxylation reaction.

Spontaneous decarboxylation contributes to spontaneity of the 2nd reaction.

Cleavage of a second ~P bond of GTP also contributes to driving synthesis of PEP.

C

C

CH 2

O O

O PO 32

C

C

CH 3

O O

O

A T P A D P + P i C

CH 2

C

C

O

O O

O

O

HC O 3

G T P G D P

CO 2

p y ru v a te o x a lo a c e ta te P E P

P y ru v a te C a rb o x y la s e P E P C a rb o x y k in a s e

Biotin has a 5-C side chain whose terminal carboxyl is in amide linkage to the e-amino group of an enzyme lysine.

The biotin & lysine side chains form a long swinging arm that allows the biotin ring to swing back & forth between 2 active sites.

Pyruvate Carboxylaseuses biotinas prosthetic group.

C HC H

H 2 C

S

C H

N H

C

H N

O

(C H 2 )4 C N H (C H 2 )4 C H

CO

N H

O

b io tin

N s u b je c t to

c a rb o x y la t io n

ly s in e

re s id u e

H 3 N+

C C O O

C H 2

C H 2

C H 2

C H 2

N H 3

H

lysine

Biotin carboxylation is catalyzed at one active site of Pyruvate Carboxylase.

ATP reacts with HCO3 to yield carboxyphosphate.

The carboxyl is transferred from this ~P intermediate to N of a ureido group of the biotin ring. Overall:

biotin + ATP + HCO3 carboxybiotin + ADP + Pi

O P O

O

O H

C O

O

carboxyphosphate

C HC H

H 2 C

S

C H

N H

C

N

O

(C H 2 )4 C N H (C H 2 )4 C H

CO

N H

O

C

O

-O

c a rb o x yb io tin

ly s in e

re s id u e

At the other active site of PyruvateCarboxylase the activated CO2 is transferred from biotin to pyruvate:

carboxybiotin+ pyruvate

biotin + oxaloacetate

View an animation.

C HC H

H 2 C

S

C H

N H

C

N

O

(C H 2 )4 C N H R

O

C

O

-OC

C

C H 3

O O

O

C

C H 2

C

C

O

O O

O

O

C HC H

H 2 C

S

C H

N H

C

H N

O

(C H 2 )4 C N H R

O

c a rb o x y b io t in

p y ru v a te

o x a lo a c e ta te

b io t in

When gluconeogenesis is active in liver, oxaloacetate is diverted to form glucose. Oxaloacetate depletion hinders acetyl CoA entry into Krebs Cycle. The increase in [acetyl CoA] activates Pyruvate Carboxylase to make oxaloacetate.

Pyruvate Carboxylase (pyruvateoxaloactate) is allosterically activated by acetyl CoA.

[Oxaloacetate] tends to be limiting for Krebs cycle.

G lu c o s e -6 -p h o s p h a ta s e

g lu c o s e -6 -P g lu c o s e

G lu c o n e o g e n e s is G ly c o ly s is

p y ru v a te

f a t ty a c id s

a c e ty l C o A k e to n e b o d ie s

o x a lo a c e ta te c i t r a te

K re b s C y c le

PEP Carboxykinase catalyzes GTP-dependent oxaloacetate PEP. It is thought to proceed in 2 steps:

Oxaloacetate is first decarboxylated to yield a pyruvate enolate anion intermediate.

Phosphate transfer from GTP then yields phosphoenolpyruvate (PEP).

C

C

C H 2

O O

O P O 32

C

C H 2

C

C

O

O O

O

O

C O 2

C

C

C H 2

O O

O

G T P G D P

o x a lo a c e ta te P E P

P E P C a rb o x y k in a s e R e a c t io n

In the bacterial enzyme, ATP is Pi donor instead of GTP.

In this crystal structure of an E. Coli PEP Carboxykinase, pyruvate is at the active site as an analog of PEP/ oxaloacetate.

Mg++

pyruvate

Mn++

ATP

PEP Carboxykinase active site ligands PDB 1AQ2

A metal ion such as Mn++ is required for the PEP Carboxykinase reaction, in addition to a Mg++ ion that binds with the nucleotide substrate at the active site.

Mn++ is thought to promote Pi transfer by interacting simultaneously with the enolate oxygen atom and an oxygen atom of the terminal phosphate of GTP or ATP.

G ly c e ra ld e h y d e -3 -p h o s p h a te

D e h y d ro g e n a s e

P h o s p h o g ly c e ra te K in a s e

E n o la s e

P E P C a rb o x y k in a s e

g ly c e ra ld e h y d e -3 -p h o s p h a te

N A D+

+ P i

N A D H + H+

1 ,3 -b is p h o s p h o g ly c e ra te

A D P

A T P

3 -p h o s p h o g ly c e ra te

P h o s p h o g ly c e ra te M u ta s e

2 -p h o s p h o g ly c e ra te

H 2 O

p h o s p h o e n o lp y ru v a te

C O 2 + G D P

G T P

o x a lo a c e ta te

P i + A D P

H C O 3

+ A T P

p y ru v a te

P y ru v a te C a rb o x y la s e

G lu c o n e o g e n e s is

Summary of Gluconeogenesis Pathway:

Gluconeogenesis enzyme names in red.

Glycolysis enzymenames in blue.

G lu c o s e -6 -p h o s p h a ta s e

F ru c to s e -1 ,6 -b is p h o s p h a ta s e

g lu c o s e G lu c o n e o g e n e s is

P i

H 2 O

g lu c o s e -6 -p h o s p h a te

P h o s p h o g lu c o s e Is o m e ra s e

f ru c to s e -6 -p h o s p h a te

P i

H 2 O

f ru c to s e -1 ,6 -b is p h o s p h a te

A ld o la s e

g ly c e ra ld e h y d e -3 -p h o s p h a te + d ih y d ro x y a c e to n e -p h o s p h a te

T r io s e p h o s p h a te

Is o m e ra s e

( c o n t in u e d )

Glycolysis & Gluconeogenesis are both spontaneous. If both pathways were simultaneously active in a cell, it would constitute a "futile cycle" that would waste energy.

Glycolysis:

glucose + 2 NAD+ + 2 ADP + 2 Pi

2 pyruvate + 2 NADH + 2 ATPGluconeogenesis:

2 pyruvate + 2 NADH + 4 ATP + 2 GTP

glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi

Questions:

1. Glycolysis yields how many ~P ?

2. Gluconeogenesis expends how many ~P ?

3. A futile cycle of both pathways would waste how many~P per cycle ?

2

6

4

To prevent the waste of a futile cycle, Glycolysis & Gluconeogenesis are reciprocally regulated.

Local Control includes reciprocal allosteric regulation by adenine nucleotides.

Phosphofructokinase (Glycolysis) is inhibited by ATP and stimulated by AMP.

Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP.

f ru c to se -6 -p h o s p h a te f ru c to se -1 ,6 -b is p h o s p h a te

P h o s p h o fru c to k in a s e

C H 2 O P O 32

O H

C H 2 O H

H

O H H

H H O

O

6

5

4 3

2

1 C H 2 O P O 32

O H

C H 2 O P O 32

H

O H H

H H O

O

6

5

4 3

2

1

A T P A D P

P i H 2 O

F ru c to s e -1 ,6 -b io s p h o s p h a ta s e

The opposite effects of adenine nucleotides on

Phosphofructokinase (Glycolysis)

Fructose-1,6-bisphosphatase (Gluconeogenesis)

insures that when cellular ATP is high (AMP would then be low), glucose is not degraded to make ATP.

When ATP is high it is more useful to the cell to store glucose as glycogen.

When ATP is low (AMP would then be high), the cell does not expend energy in synthesizing glucose.

Global Control in liver cells includes reciprocal effects of a cyclic AMP cascade, triggered by the hormone glucagon when blood glucose is low.

Phosphorylation of enzymes & regulatory proteins in liver by Protein Kinase A (cAMP Dependent Protein Kinase) results in

inhibition of glycolysis

stimulation of gluconeogenesis,

making glucose available for release to the blood.

Enzymes relevant to these pathways that are phosphorylated by Protein Kinase A include:

Pyruvate Kinase, a glycolysis enzyme that is inhibited when phosphorylated.

CREB (cAMP response element binding protein) which activates, through other factors, transcription of the gene for PEP Carboxykinase, leading to increased gluconeogenesis.

A bi-functional enzyme that makes and degrades an allosteric regulator, fructose-2,6-bisphosphate.

Recall that Phosphofructokinase, the rate-limiting step of Glycolysis, is allosterically inhibited by ATP.

At high concentration, ATP binds at a low-affinity regulatory site, promoting the tense conformation.

0

1 0

2 0

3 0

4 0

5 0

6 0

0 0 .5 1 1 .5 2

[F ru c to s e -6 -p h o s p h a te ] m M

PF

K A

cti

vit

y

h ig h [A T P ]

lo w [A T P ]

Sigmoidal dependence of reaction rate on [fructose-6-phosphate] is observed at high [ATP].

Fructose-2,6-bisphosphate promotes the relaxed state, activating Phosphofructokinase even at high [ATP].

Thus activation by fructose-2,6-bisphosphate, whose concentration fluctuates in response to external hormonal signals, supersedes local control by [ATP].

0

1 0

2 0

3 0

4 0

5 0

6 0

0 0 .5 1 1 .5 2

[F ru c to s e -6 -p h o s p h a te ] m M

PF

K A

cti

vit

y

h ig h [A T P ]

lo w [A T P ] PFK activity in the presence of the globally controlledallosteric regulator fructose-2,6-bisphosphate is similar to that at low ATP.

Phosphofructokinase-2 (PFK2) domain catalyzes:Fructose-6-phosphate + ATP fructose-2,6-bisphosphate + ADP

Fructose-Biophosphatase-2 (FBPase2) domain catalyzes:Fructose-2,6-bisphosphate + H2O fructose-6-phosphate + Pi

Bifunctional PFK2/FBPase2 assembles into a homodimer.

P F K 2 /F B P a s e 2 h o m o d im e r P D B

2 B IF

P F K -2

d o m a in

F B P a s e -2

d o m a in

w ith b o u n d

f ru c to s e -6 -P

in a c t iv e s i te

The allosteric regulator fructose-2,6-bisphosphateis synthesized & degraded by a bi-functional enzymethat includes 2 catalytic domains:

Adjacent to the PFK-2 domain in each copy of the liver enzyme is a regulatory domain subject to phosphorylation by cAMP-dependent Protein Kinase.

Which catalytic domains of the enzyme are active depends on whether the regulatory domains are phosphorylated.

P F K 2 /F B P a s e 2 h o m o d im e r P D B

2 B IF

P F K -2

d o m a in

F B P a s e -2

d o m a in

w ith b o u n d

f ru c to s e -6 -P

in a c t iv e s i te

cAMP-dependent phosphorylation of the bi-functional enzyme activates FBPase2 and inhibits PFK2.

[Fructose-2,6-bisphosphate] thus decreases in liver cells in response to a cAMP signal cascade, activated by glucagon when blood glucose is low.

( a c t iv e a s P h o s p h o f ru c to k in a s e -2 )

E n z -O H

A T P A D P

f ru c to s e -6 -P f ru c to s e -2 ,6 -b is P

P i

E n z -O -P O 3

2

( a c t iv e a s F ru c to s e -B is p h o s p h a ta s e -2 )

View an animation.

Glycolysis slows because fructose-2,6-bisphosphate is not available to activate Phosphofructokinase.

Gluconeogenesis increases because of the decreased concentration of fructose-2,6-bisphosphate, which would otherwise inhibit the gluconeogenesis enzyme Fructose-1,6-bisphosphatase.

( a c t iv e a s P h o s p h o f ru c to k in a s e -2 )

E n z -O H

A T P A D P

f ru c to s e -6 -P f ru c to s e -2 ,6 -b is P

P i

E n z -O -P O 3

2

( a c t iv e a s F ru c to s e -B is p h o s p h a ta s e -2 )

Downstream effects of the cAMP cascade:

Summary of effects of glucagon-cAMP cascade in liver:

Gluconeogenesis is stimulated.

Glycolysis is inhibited.

Glycogen breakdown is stimulated.

Glycogen synthesis is inhibited.

Free glucose is formed for release to the blood.

G ly c o g e n P y ru v a te

G lu c o n e o g e n e s is

G lu c o s e -1 -P G lu c o s e -6 -P G lu c o s e + P i

G lu c o s e -6 -P a s e

G ly c o ly s is

P a th w a y

X

X

Energetics of Gluconeogenesisfigure 13-1

• Pyruvate Carboxylase– 2 ATPs

• PEP Carboxykinase– 2 GTPs

• 3-P-glycerate kinase– 2 ATPs

• Glyceraldehyde-3-P dehydrogenase– 2NADH

Pag

e 8

51

Glyoxylate cycle

• Hans Kornberg and Neil Madsen

• The glyoxylate cycle results in the net conversion of two acetyl-CoA to succinate instead of 4 CO2 in citric acid cycle.

• Succinate is transferred to mitochondrion where it can be converted to OAA (TCA)

• Can go to cytosol where it is converted to oxaloacetate for gluconeogenesis.

Net reaction

2Ac-CoA + 2NAD+ + FAD OAA + 2CoA + 2NADH +FADH2 + 2H+

Plants are able to convert fatty acids to glucose through this pathway

Glyoxylate cycle

• The glyoxylate cycle results in the net conversion of two acetyl-CoA to succinate instead of 4 CO2 in citric acid cycle.

• Succinate is transferred to mitochondrion where it can be converted to OAA (TCA)

• Can go to cytosol where it is converted to oxaloacetate for gluconeogenesis.

Net reaction

2Ac-CoA + 2NAD+ + FAD OAA + 2CoA + 2NADH +FADH2 + 2H+

Plants are able to convert fatty acids to glucose through this pathway

Reference

• cronus.uwindsor.ca/units/biochem/web/.../Citric%20acid%20cycle.ppt

• www.elmhurst.edu/~chm/onlcourse/CHM103/Rx24citricacidcycle.ppt

• www.philadelphia.edu.jo/courses/biology/The%20Krebs%20Cycle.ppt

• www.uh.edu/sibs/faculty/glegge/lecture_23a.ppt• www2.fiu.edu/~bch3033/Handouts/Lh6Ch16TCA.ppt•