Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431

Biochemistry

Fall 2008

Lecture Notes: 19 November

© R. Paselk 2008
 
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Kreb's TCA Cycle, cont.

Mainline Sequence reactions

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

structural diagram of the "Mainline sequence" of 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)*

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

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Last modified 19 November 2008