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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Solutions to problem set 3

1.

malate + NAD+ oxaloacetate + NADH + H+; = + 29.7 kJ/mol

maximum oxaloacetate

2.

If oxaloacetate is supplied from another source causing the level to rise above 6.3 x 10-7 M, malate dehydrogenase will reverse and malate will acumulate to higher levels. This may lead to malate export, especially if malate is being consumed in the cytoplasm. Citrate synthase may also consume some of the extra oxaloacetate, but this enzyme may be limited by its capacity since it is not quite at equilibrium.

oxaloacetate + acetyl-CoA citrate + HSCoA; = - 34.2 kJ/mol

minimum oxaloacetate

Since the minimum [oxaloacetate] needed to make citrate synthase run is much less than the maximum put out by malate dehydrogenase, the reaction sequence can still proceed even though the absolute steady state concentration of oxaloacetate is so low.

3.

Assuming transport equilibrium, we can take 10 mM malate as the cytoplasmic concentration. In the cytoplasm, the relative [NADH] / [NAD+] ratio is much lower, favouring oxidation.

cytoplasmic oxaloacetate
This allows a much higher level of oxaloacetate to accumulate than is available in the mitochondrion, helping the PEP carboxykinase if gluconeogenesis is active.

4.

oxaloacetate pyruvate + CO2 = - 31.8 kJ/mol
GTP + H2O GDP + Pi = - 30.3 kJ/mol
pyruvate + Pi PEP + H2O = + 61.9 kJ
oxaloacetate + GTP PEP + GDP + CO2 = - 0.2 kJ/mol

For CO2 as a product: CO2 effect

This contribution is due to the low content of CO2 in the atmospher, and is in addition to any negative contribution to due to resonance stabilization.

If CO2 is a reactant, the partial pressure term becomes a reciprocal, and a positive contribution to will result.

The [products] / [ reactants] term in the equation should technically use activities rather than concentrations. At a first approximation, which is OK for low pressures and concentrations, activity = [actual] / [standard]. Since the [standard] is 1 mol/L or 1 atm, we end up with the numerical values of concentrations of solutions or partial pressure of gases in the equation.

5.

malate + NAD+ oxaloacetate + NADH + H+; = + 29.7 kJ/mol
oxaloacetate pyruvate + CO2 = - 31.8 kJ/mol
malate + NADP+pyruvate + NADPH + CO2 = - 2.1 kJ/mol

The low negative has led some authors to suggest that this reaction is easily reversible - a dangerous assumption if no effort is made to consider . This reaction has two reactants and three products, and this will skew the final towards negative values. The CO2 product will contribute -25.8 kJ to . Taking typical cytoplasmic pyruvate levels of 51 µM (lecture 2) and [NADPH] / [NADP+] = 50, we can estimate the maximum [malate]:

malic enzyme calculation

It should be clear that the resulting [malate] is ridiculously low, so that it would result in [oxaloacetate] < 10-12 M. Hence malic enzyme should not be considered as readily reversible.

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