1. Outline the steps required to get one molecule of acetyl CoA from inside themitochondrion, where pyruvate dehydrogenase is located, to the cytoplasm.

Acetyl-CoA is made in the mitochondrion by pyruvate dehydrogenase, by β-oxidation and byamino acid oxidation. However there is no transporter for acetyl-CoA in the mitochondrialmembrane, so acetyl CoA has to be transported indirectly.(M) = in the mitochondrion; (C) = in the cytoplasm

citrate synthase (mitochondrial)Acetyl-CoA(M) + oxaloacetate (M) →→ citrate(M) + HSCoA(M)

Translocation across membrane:citrate translocator (mitochondrial membrane)

citrate(M) + malate(C) →→ citrate(C) + malate(M)

citrate lyase (cytoplasmic)Citrate(C) + HSCoA(C) + ATP(C) →→ acetyl-CoA(C) + oxaloacetate (C) + ADP(C) + Pi(C)

malate dehydrogenase (cytoplasmic)Oxaloacetate (C) + NADH(C) + H+ →→ malate(C) + NAD+

(C)

For malate translocation, see abovemalate dehydrogenase (mitochondrial)

malate(M) + NAD+(M) → → oxaloacetate (M) + NADH(M) + H+

NADH is also not transported directly, so this process helps use up cytoplasmic NADH, whileproducing more mitochondrial NADH for use in oxidative phosphorylation.

2. Write down and sum the equations for citrate synthase (Intro Biochem, TCA cycle) andcitrate lyase.

citrate synthaseAcetyl-CoA + oxaloacetate →→ citrate + HSCoA

citrate lyaseCitrate + HSCoA + ATP → → acetyl-CoA + oxaloacetate + ADP + Pi

ATP → → ADP + Pi

What prevents the combination of citrate synthase and citrate lyase from being a futilecycle?

A futile cycle may occur when two opposed reactions (product of one is reactant of the other)differ in that ATP hydrolysis in one direction is not balanced out by ATP synthesis in the otherdirection. The net sum of the two reactions has ATP hydrolysis as the only outcome, e.g.

Fructose-6-phosphate + ATP → → Fructose-1,6-bisphosphate + ADPFructose-1,6-bisphosphate + H2O →→ Fructose-6-phosphate + Pi

ATP → → ADP + Pi

Chem*3560 Question set 7 Feb 23-27, 2004

The two different reactions are necessary to ensure that each progresses in the designateddirection, whereas a true reversal of the phosphofructokinase reaction is disallowed by the freeenergy change under cellular conditions (∆G = –26 kJ/mol).

The difference with citrate synthase and citrate lyase reaction is that the acetyl-CoA product ofthe citrate lyase reaction is not the reactant of citrate synthase, because acetyl CoA substratereacts in the mitchondrion, but the acetyl-CoA product is generated in the cytoplasm.Oxaloacetate eventually returns to the mitochondrion (see question 1), so the true sum to thetwo equations is:Acetyl-CoA(mito) + oxaloacetate →→ citrate + HSCoACitrate + HSCoA + ATP →→ acetyl-CoA(cyto) + oxaloacetate + ADP + Pi

Acetyl-CoA(mito) ATP →→ acetyl-CoA(cyto) + ADP + Pi

The ATP hydrolysis in this process is not futile, but contributes chemical driving force to theexport of acetyl CoA from the mitochondrion.

3. If fatty acid biosynthesis is the reverse of β-oxidation, a four step cycle, why is fatty acidbiosynthesis a six step cycle?

In most organisms, fatty acid synthase is a closely associated complex of seven catalyticcentres surrounding acyl carrier protein (ACP) which carries a long pantetheine armterminating in the HS- group that bonds to the substrate. In bacteria, the complex consists ofindependent polypeptides held together by non covalent forces, one for each catalyticcomponent plus ACP. In animals, the fatty acid synthase components are strung out as multipledomains on a single polypeptide chain. The functional enzyme is a dimer of two identical chains.

In fatty acid biosynthesis, the growing acyl chain becomes covalently bonded to the enzyme'sACP and its pantetheine chain. This increases fatty acid synthase's efficiency, since thesubstrate remains in close range of the six catalytic centres instead of disappearing into thesurroundings, and having to return to the enzyme by random diffusion. The first four reactions ofthe fatty acid cycle backtrack through the four β-oxidation steps.

1) Ketoacyl-ACP synthase reverses the thiolase reaction2) Ketoacyl-ACP reductase reverses the hydroacyl-CoA dehydrogenase step3) Hydroxyacyl-ACP dehydratase reverses enoyl-CoA hydratase4) Enoyl-ACP reductase reverses acyl-CoA dehydrogenase

and this gives:

CH3-CH2-CH2-CO-S-ACP, which will be elongated by reaction with malonyl-S-CoA ↑ ↑

The malonyl radical reacts here, not at the CH3- end, which is chemically inert.

Chem*3560 Question set 7 Feb 23-27, 2004

If CH3-CH2-CH2-CO-S-ACP reacted directly with malonyl-S-CoA, the product would bethe ketoacyl-S-CoA, leaving HS-ACP vacant. The ketoacyl-S-CoA could then be lost fromfatty acid synthase and would have to diffuse back to the enzyme, making the process lessefficient. The extra two steps of acyl transferase and malonyl transferase overcome theproblem:

acyl transferaseCH3-CH2-CH2-CO-S-ACP + HS-Enz →→ CH3-CH2-CH2-CO-S-Enz + HS-ACP

where HS-Enz is a specific Cys sidechain in the ketoacyl-ACP synthase catalytic site. Thiskeeps the growing chain covalently bonded to the fatty acid synthase, right in the catalytic sitewhere it will next react, in ketoacyl-ACP synthase.

malonyl transferasemalonyl-S-CoA + HS-ACP →→ malonyl-S-ACP + HSCoA (free to leave)

This covalently bonds the incoming malonyl to the fatty acid synthase ACP.Finally, ketoacyl-ACP synthase catalyzes the joining of the two substrates:

ketoacyl-S-ACP synthaseCH3-CH2-CH2-CO-S-Enz + + malonyl-S-ACP → → CH3-CH2-CH2-CO-CH2-CO-S-ACP

+ HS-Enz + CO2

This leaves product on the ACP, ready to continue the next reaction cycle.

4. If cytoplasmic malic enzyme converts malate to pyruvate in order to generate NADPH,how does the TCA cycle in the mitochondrion get more oxaloacetate to sustain the TCAcycle?

When there is sufficient glucose in the cell to supply NADPH by the pentose phosphate cycle,oxaloacetate returns to the mitochondrion by the following processes:(M) mitochondrial (C) cytoplasmic

citrate lyase (cytoplasmic)Citrate(C) + HSCoA(C) + ATP(C) →→ acetyl-CoA(C) + oxaloacetate (C) + ADP(C) + Pi(C)

malate dehydrogenase (cytoplasmic)Oxaloacetate (C) + NADH(C) + H+ →→ malate(C) + NAD+

(C)

citrate translocator (mitochondrial membrane)citrate(M) + malate(C) →→ citrate(C) + malate(M)

malate dehydrogenase (mitochondrial)malate(M) + NAD+

(M) → → oxaloacetate (M) + NADH(M) + H+ regenerates citrate (M)

Chem*3560 Question set 7 Feb 23-27, 2004

If acetyl-CoA is bing produced from amino acids instead of from glucose, there may be nosubstrate to make NADPH by the pentose phosphate pathways. In this case, malate canprovide NADPH:

malate dehydrogenase (decarboxylating)malate(C) + NADP+

(C) → → pyruvate(C) + CO2 + NADPH(C) + H+

but this poses the question of what supplies oxaloacetate to keep the TCA cycle going.

Pyruvate enters the mitochondrion through a specific transporter, and is converted tooxaloacetate by pyruvate carboxylase:

pyruvate carboxylasepyruvate(M) + CO2 + ATP(M) →→ oxaloacetate (M) + ADP(M) + Pi(M)

This pathway costs an extra ATP to make NADPH in place of the the NADH produced in thepathway that uses malate transport.

citrate citrate ↓ ATP used ↓ ATP usedoxaloacetate + acetyl-CoA oxaloacetate + acetyl-CoA ↓ NADH used ↓ NADH usedmalate malate ↓ transport ↓ NADPH mademalate pyruvate + CO2

↓ NADH made ↓ transportoxaloacetate pyruvate

↓ ATP usedoxaloacetate

NADPH is not a substrate for oxidative phosphorylation, so deriving its energy equivalence inunits of ATP is not straightforward. However the process shown here is one example of severalthat suggests that in terms of energy equivalence,NADPH = NADH + ATP.

The extra energy content of NADPH is the consequence of the high ratio [NADPH] / [NADP+].

5. What effects other than fatty acid biosynthesis may be expected from the presence ofcitrate in the cytoplasm?

Cytoplasmic citrate is a negative allosteric regulator of phosphofructokinase 1 and apositive regulator of fructose-1,6-bishosphatase, so would normally shut down glycolysisand activate gluconeogenesis. Insulin can stimulate phosphofructokinase 2 in liver, and thefructose-2,6-bisphosphate produced would override the effect of citrate. This allows glycolysisto continue feeding substrate to fatty acid biosynthesis in the liver.

Chem*3560 Question set 7 Feb 23-27, 2004