Chem*4520 Metabolic Processes

Fall Semester 2000

Modified August 2000

schematic view of the enzyme citrate synthase,
with bound acetyl-CoA analog in green

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Lecture 6:

Gluconeogenesis: turning the thermodynamics around.

Fri Sept 22Voet Chapter 21, pp.600-606,, 657-658.
Mathews, Van Holde: Chapter 16 pp. 561-572.
Stryer Chapter 22, pp. 569-577.

Gluconeogenesis

Purpose: gluconeogenesis permits the replenishment of glucose from metabolic sources other than carbohydrates.
- for polysaccharide biosynthesis in plants or bacteria
- in animals certain organs require glucose (brain, some skeletal muscles, red blood cells) and this is provided by gluconeogenesis in liver and kidney
- recycling of lactate after anaerobic activity.

Substrate for gluconeogenesis comes from:
- lactate, recycled after anaerobic activity
- amino acid breakdown
- plants and bacteria which have the glyoxylate cycle can use acetyl CoA from fat and oil breakdown. Animals can't convert fats to carbohydrate.

Reactions of glycolysis which are close to equilibrium are easily reversed for gluconeogenesis. Small changes in concentrations of glycolysis pathway intermediates are sufficient to allow net flux in reverse.

Three reactions of glycolysis are not easily reversed and must be bypassed for gluconeogenesis.

kinases are bypassed by phosphatases

Hexokinase and phosphofructokinase involve phosphate transfer from ATP to substrate, and these steps are easily bypassed by specific phosphatases, which hydrolyse the sugar phosphate without recovery of ATP.

The phosphatase reaction is a distinct reaction, not a true reversal of the kinase reaction, so both directions can have negative free energy change.


Conversion of pyruvate to PEP

This is much tricker. PEP has a very large free energy of hydrolysis ( = -61.9 kJ/mol), so simple phosphate transfer from ATP is ruled out. Essentially, synthesis of the PEP phosphate is worth two ATP high energy phosphates, and one way or another 2 ATP are consumed per PEP made.

In bacteria and plants (Voet p.657-8, Mathews p.623), pyruvate dikinase catalyzes

pyruvate + ATP + Pi PEP + AMP + PPi = +28.7 kJ/mol

This is supported by the action of pyrophosphatase (PPi = HP2O73-, pyrophosphate).

PPi + H2O 2 Pi = - 33.4 kJ/mol.

The two reactions sum to = -4.7 kJ/mol.

The reactions are coupled by the low steady state concentration of PPi. Pyrophosphatase is a highly efficient enzyme and keeps the [PPi] down to about 10-11 M. This low concentration of product for the dikinase reaction results in negative despite the large positive .

Although the dikinase only consumes one ATP, a second ATP is used to restore AMP to the level of ADP, which is the normal "currency" for rephosphorylation.

adenylate kinase
ATP + AMP 2 ADP


A more complex process occurs in animals

In animals, the main pools of pyruvate are in the mitochondria, but gluconeogenesis takes place in the cytoplasm. Since it makes no sense to derive pyruvate from glucose to convert it back to glucose, pyruvate is generated either by oxidation of lactate or by degradation of small amino acids Ala, Ser, Gly, Cys and Thr.

The mitochondrial enzyme pyruvate carboxylase converts pyruvate to oxaloacetate:
pyruvate carboxylase
CH3-CO-CO2- + CO2
pyruvate
biotin
-O2C-CH2-CO-CO2-
oxaloacetate

Oxaloacetate may also be derived from amino acids that breakdown into TCA cycle intermediates:
Asp, Asnoxaloacetate directly
Glu, Gln, Arg, Pro, His-ketoglutarate
Ile, Met, Val
(via propionyl-CoA)
succinate
Phe, Tyrfumarate

These breakdown processes occur in mitochondria. There is no transporter for oxaloacetate to cross the mitochondrial membrane, so excess oxaloacetate is reduced back to malate in the mitochondria, transported to the cytoplasm and then reoxidized to oxaloacetate:
oxaloacetate
NADHNAD+
malate antiporter malate
NAD+NADH
oxaloacetate
mitochondrialmitochondrialmembrane
transporter
cytoplasmiccytoplasmic

Although this may seem convoluted, it has two benefits:

In the cytoplasm, oxaloacetate accepts phosphate from GTP yielding PEP in a reaction coupled to decarboxylation:
PEP carboxykinase
-O2C-CH2-CO-CO2-
oxaloacetate
CO2 + PEP
GTPGDP

The driving force for this reaction is the decarboxylation. The reaction series

R-CHOR-CO2-R-H + O=C=O

shows a progressive increase in resonance stabilization going from aldehyde to carboxylate and finally to carbon dioxide. Therefore either aldehyde oxidations or decarboxylations can make significant contributions to negative .

The contribution from decarboxylation of oxaloacetate is easily estimated:
H+ +oxaloacetate2-pyruvate- + CO2
- 39.9 kJ/mol- 797.2 kJ/mol- 474.5 kJ/mol- 394.4 kJ/mol
f values shown below each reactant and product give net = -31.8 kJ/mol.

The decarboxylation contributes an energy equivalent to one high energy phosphate, so that the GTP can transfer a single phosphate to form PEP. The previous carboxylation of pyruvate (costing 1 ATP) has stored energy as an easily eliminated carboxylate group; the stored bond energy assists the phosphate transfer from the GTP.

Recall that the high energy of phosphoenol pyruvate hydrolysis is partly derived from the isomerization of the enolate anion to a carbonyl in pyruvate.

decarboxylation of oxaloacetate

The decarboxylation mechanism gives rise to the enolate anion as an intermediate, so that the phosphate transfer does not face as severe an energy barrier as it would if pyruvate is the substrate.


Regulation of key enzymes

(Voet pp. 472-474,p.605; Matthews p. 570; Stryer p.575)

There is some variation in different tissues and organisms, but a common theme of regulation is that ATP inhibits and AMP activates key glycolysis enzymes, especially phosphofructokinase, while ATP activates and AMP inhibits the gluconeogenesis enzyme fructose-1,6-bisphosphatase. Early steps of gluconeogenesis are controlled by the low level of available oxaloacetate when metabolism is directed towards ATP production.

Why AMP?

AMP levels in the cell are governed by the adenylate kinase equilibrium:

ATP + AMP 2 ADPKeq= 2.3.

myokinase equilibrium[AMP] depends on the square of [ADP], so doubling [ADP] gives at least a fourfold increase in [AMP]. In addition, there's a reciprocal dependence on [ATP]. Since a decrease in [ATP] usually means an increase in [ADP], a small change in [ATP] can result in a huge change in [AMP]. Hence [AMP] levels are a sensitive indicator of the energy status of a cell.

Substrate cycling

If an enzyme pair catalyzing opposed reactions such as phosphofructokinase and fructose-1,6-phosphatase are simultaneously active, there will be a reaction cycle resulting in net hydrolysis of ATP. This is known as a substrate cylce, and can be an important factor in regulation.

metabolic controlIn these examples the effect of activation or inhibition by AMP and ATP are illustrated.

In a), the system is idling under moderately high [ATP] levels. Lowish phosphofructokinase (PFK) activity is counteracted by moderate activity of fructose bisphosphatase (FBPase), to give net flux of 1 unit.

In b), AMP activates PFK and inhibits FBPase by a factor of 9 each, a reasonable amount for an allosteric enzyme. However flux increases from 1 to 89 units.

c) may represent the state in liver, where FBPase can be activated by ATP, or with very high [ATP] levels in muscle tissue. The activity of PFK has dropped below FBPase, and a modest 3.3 fold activation of FBPase is translated into a significant net flux in the gluconeogenesis direction.

Substrate cycles consume small amounts of ATP, but give a broader range of control over metabolic rates for small changes in effector concentration.


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