Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2010

Lecture Notes: 7 April

© R. Paselk 2006
 
PREVIOUS  

NEXT

Kreb's TCA Cycle

The Tricarboxylic acid cycle is in many ways the central pathway of metabolism, both catabolically and anabolically: it is involved in the breakdown and synthesis of a variety of compounds. Right now we want to focus on its catabolic role in aerobic catabolism: the oxidative breakdown of the acetyl group of acetyl CoA. In this instance we can consider the entire cycle to be a catalyst for this breakdown.

The problem is that the C-C bond of the acetyl group is chemically very resistant. Recall that in organic chemistry generally get C-C bond cleavages at alpha-beta bonds to carbonyl carbons, but with the acetyl group there is no beta carbon. So the TCA Cycle creates an alpha-beta bond by first attaching the acetyl group onto a carrier molecule, oxaloacetate.

Let's look at an overview of the Kreb's TCA Cycle. First condense the acetyl group with a four carbon carrier to get a six carbon tri-acid. This is then rearranged and oxidized with loss of carbon dioxide to give a five carbon di-acid ketol very similar to pyruvate in structure. An irreversible DH Complex then creates a four carbon CoA derivative with the release of a second carbon dioxide. At this point it appears that acetyl has been released as carbon dioxide, however, the carrier has been reduced, and modified. A series of reactions now regenerates the original carrier.

The first reaction of the cycle is an aldol condensation catalyzed by

Citrate synthase

Structural diagram for citrate synthase reaction

Note that the enzyme catalyst enables the coupling of two chemically independent reactions: the aldol condensation (with free energy change of about zero) to the very favorable hydrolysis of the CoA thiol ester bond which drives the overall reaction far towards product. Essentially, we have used an ATP's worth of energy to drive the reaction to completion.

Aconitase

Unfortunately the resulting citrate is a tertiary alcohol which cannot be readily oxidized. Aconitase catalyzes the rearrangement of citrate to give an oxidizable secondary alcohol. This reaction involves an elimination/addition sequence, catalyzed by an iron-sulfur cluster (Fe4S4), with an alkene intermediate, cis- Aconitate:

Aconitase reaction structures

We have now converted the 3° alcohol, citrate, into an oxidizable 2° alcohol, isocitrate. The next reaction is the first oxidation of the TCA Cycle.

Isocitrate DH

The isocitrate alcohol can now be oxidized with NAD+ by Isocitrate DH to give an enzyme bound intermediate. The intermediate has a carboxyl group beta to a carbonyl carbon, so it has an excellent leaving group, CO2, attached to a stabilized carbanion. Thus it immediately rearranges to lose carbon dioxide:

Isocitrate DH reaction including structures

Ketoglutarate DH Complex

The resulting 2-oxo-glutarate (-ketoglutarate) looks just like pyruvate with an R-group attached to the -carbon, so it is broken down by a DH Complex, the -Ketoglutarate DH Complex, just as pyruvate was. This gives succinyl-CoA and releases a second carbon dioxide. Note that at this point two carbons have been released, so formally, we have released the two carbons of Acetyl-CoA (Though neither of them came from the acetyl CoA we added)! We have also produced two NADH's (4 NADH/Glucose) which will result in the production of 5 ATP's (or: 4 x 2.5 ATP/NADH= 10 ATP's/Glucose). However, we have not regenerated the carrier. The remainder of the cycle is involved in this regeneration. (Note in the first four reactions two carbons have been lost as CO2 - as many carbons have been lost as were picked up with acetate. In a sense the rest of the cycle is regenerating our carrier - oxalacetate!)

Succinyl-CoA Synthetase

Succinyl-CoA, like acetyl-CoA, has a high-energy bond. However in this case the energy will be captured, using Succinyl-CoA Synthetase, to give a GTP which is energetically equivalent to an ATP (2 ATP's/Glucose). The mechanism of this reaction first involves the phosphorolysis by inorganic phosphate of the thiol ester bond to give a phosphoric-carboxylic mixed acid anhydride, followed by formation of a phosphorylated enzyme and finally transfer of the phosphate onto GDP.

Mainline Sequence

The reactions beginning with succinate are representative of a common pattern, the "Mainline Sequence," seen repeatedly in biochemical pathways.

generalized diagram for mainline sequence reactions

Succinate DH

First, Succinate DH, an inner-mitochondrial membrane-bound enzyme and member of the mitochondrial electron transport system (ETS), oxidizes succinate to fumerate. This reaction uses the stronger oxidizer FAD as an oxidizing agent because of the added difficulty in oxidizing an alkane to an alkene. As a consequence of using this more powerful oxidizing agent, less ATP energy can be captured in oxidizing the resulting FADH2 with oxygen (FAD is closer to oxygen in its oxidation potential). One and one-half ATP equivalents are obtained in this reaction (or: 2 x 1.5 ATP/FADH2= 3 ATP/Glucose).

Fumerase

The resulting alkene, Fumerate, is not readily oxidized. However, if water is added across the double bond an alcohol results which can be oxidized. Thus Fumerase catalyses a hydration reaction to give malate.

Malate DH

Finally, Malate DH catalyzes the dehydrogenation of malate to regenerate the original carrier, oxaloacetate, and finish the cycle. In addition another NADH is formed (and 2 x 2.5 ATP/NADH= 5 ATP/Glucose).

For the entire cycle we then have the production of 10 ATP/acetyl-CoA or 20 ATP/Glucose. The aerobic catabolism of glucose can then give a maximum total of 32 ATP/glucose as summarized in the Table:

* In some tissues (insect flight muscle, fast twitch muscle) the reducing equivalents of NADH must be pumped against a gradient at a cost of 1 ATP (it is used to make FADH2).

 Reaction
Energy Product

factor
ATP Equivalents
(@2.5 ATP/NAD)
ATP Equivalents
(@3 ATP/NAD)
Glycolysis
 Hexokinase
ADP

1 x -1

- 1
-1 
 PFK
ADP

1 x-1

- 1
 -1
 GA-3-P DH
NADH

2 x 2.5 (1.5)*

5 (3)*
6 (4)*
PGA Kinase
ATP

2 x1

2
 2
Pyruvate Kinase
ATP

2 x 1

2
 2
 Pyruvate DH Complex & Kreb's Cycle
Pyruvate DH Complex
NADH

2 x 2.5

5
 6
Isocitrate DH
NADH

2 x 2.5

5
2-oxoglutarate DH Complex
NADH

2 x 2.5

5
 6
Succinyl-CoA Synthetase
GTP

2 x 1

2
Succinate DH
FADH2

2 x 1.5

3
 4
Malate DH
NADH

2 x 2.5

5
 6

  TOTAL=

32 (30)*
 38 (36)*

Note that because all catalysts (oxaloacetate, enzymes etc.) must be regenerated in looking at the overall operation of the cycle, only the acetyl group of acetyl-CoA can be oxidized completely. Some intermediates, such as citrate, can be partially oxidized, but Kreb's cycle intermediate catabolism requires leaving the cycle at oxaloacetate and then returning as acetyl-CoA. Note that this requires leaving the mitochondria for some reactions, and since the extremely low concentrations of oxaloacetate don't allow its efficient transport across the mitochondrial membrane (the Km of the carrier is much higher than [oxaloacetate]), malate is the species which actually leaves the mitochondria.

Regulation of the TCA Cycle

In order to understand the regulation of the TCA cycle we need to look at the DeltaG values for the various reactions and the kinetic properties of the enzymes. Values for the non-equilibrium reactions are tabulated below:

Data from E. A. Newsholme and A. R. Leach Biochemistry for the Medical Sciences. John Wiley & Sons, New York (1983) pp 101 & 110.

 Enzyme Substrate Substrate Conc. (mM)  Km (mM) DeltaG (kJ/mol)  Effectors
 Citrate synthase acetyl-CoA 100-600 5-10 -53.9
Succinyl CoA (-), ATP (-), NADH (-)
oxaloacetate 1-10 5-10
 Isocitrate DH (NAD+) isocitrate 150-700 50-200 -17.5
 Ca2+ (+), ATP (-), ADP(+), NADH (-)
 2-Oxoglutarate DH 2-oxoglutarate 600-5900 60-200 -43.9
 Ca2+ (+), Succinyl CoA (-), NADH (-)

Note that normally the concentrations of ATP, ADP, NAD+, and NADH are relatively constant in the mitosol and are thus unlikely to be very effective as allosteric regulators under most circumstances. On the other hand the availability of NAD+ and FAD as substrate will affect the rate not only of the reactions in the table, but also the near-equilibrium dehydrogenases. Note that NAD+ availability in turn is determined by the activity of the electron transport system, whose activity is closely coupled to the availability of ADP. Thus high [ATP] will slow the TCA cycle since high [ATP] means low [ADP], which will slow the ETS resulting in low [NAD+]!

In muscle, Ca2+ does show significant changes in concentration in the mitosol (recall that an increase in [Ca2+] concentration initiates muscle concentraction). Succinyl CoA will also show significant concentration changes under differing conditions and can thus also serve as an effective regulator, indicating carbon status in the second half of the cycle.


Pathway Diagrams

C438 Home

Lecture Notes

Last modified 7 April 2010