Chem*4520 Metabolic Processes Fall Semester 2000 Modified August 2000
schematic view of the enzyme citrate synthase, with bound acetyl-CoA analog in green	Department of Chemistry and Biochemistry Home Page Back to Chem*4520 Home Page

Solutions to problem set 4 Oct 2-6

1.

Aspartate antiport is balanced by glutamic acid; mitochondrial membrane potential favours aspartate export. Since the glyoxysome does not have the same membrane potential as mitochondrion, there is no problem with the reversal of antiport at the glyoxysome. Glutamic acid acts as NH2- donor for mitochondrial transaminase so that oxaloacetate can be converted to aspartate. The excess -ketoglutarate produced in the mitochondrion can then act as antiport exchange substrate for succinate. Since as much -ketoglutarate is produced as is exported, there is no net change in mitochondrial -ketoglutarate levels. -ketoglutarate arriving in the glyoxysome accepts NH2- from aspartate via the glyoxysomal transaminase.

2.

pyruvate-proton symporter

pyruvate^-_out + H⁺_outpyruvate^-_in + H⁺_in = 0 for electroneutral transport.

This is not electrogenic transport since the net charge of pyruvate^- + H⁺ is zero, nor is this active transport driven by direct coupling to redox or ATP hydrolysis. Pyruvate is driven in solely by the concentration gradient of H⁺ which exists at the mitochondrial inner membrane because of oxidative phosphorylation.

If pH = 0.5, [H⁺]_in/ [H⁺]_out = 0.32.
As the system approaches equilibrium, tends to zero.

Note that Mathews describes the mitochondrial pyruvate transporter as antiport with OH^- rather than symport with H⁺. This is a relic of older textbooks, e.g. the 1975 Lehninger. It's actually difficult to distinguish OH^- antiport from symport with H⁺, because any H⁺ gradient must be complemented by an opposite OH^- gradient, and the mathematical result will be the same. However, current consensus suggests that the symport is correct.

3.

tricarboxylate antiporter

citrate^2-_in + malate^2-_outcitrate^2-_out + malate^2-_in = 0.

Note that the antiport involves citrate^2- rather than citrate^3- which is the predominant form at pH > 6.5.

Thus the antiport is equation is mathematically equivalent to:

citrate^3-_in + H⁺_in + malate^2-_outcitrate^3-_out + H⁺_out + malate^2-_in

and is dependent on the H⁺ gradient. It's not at all certain which version of the two equations represents the actual antiport. Note that it's not necessary to know the pKa.

a)

b)

Note that significant export to the cytoplasm is dependent on high levels of [citrate] in the mitochondria, and adequate [malate] in the cytoplasm to return.

c) If the H⁺ gradient played no role, case a) would give 510 然, and case b) would give 5.1 mM cytoplasmic citrate. The antiport would be too "leaky" for citrate unless the H⁺ gradient acted to retain most of the citrate.

d) First calculate the equilibrium constant:

If [citrate] = 160 然, [isocitrate] = 0.075 x 160 然 = 12 然

If [citrate] = 1.6 mM, [isocitrate] = 120 然

The isocitrate level in cytoplasm is quite low, and this makes the cytoplasmic NADP⁺ dependent isocitrate dehydrogenase less significant in eukaryotes as a source of NADPH. In bacteria, where there is no compartmental separation, [citrate] and [isocitrate] levels may be high enough for NADP⁺ dependent isocitrate dehydrogenase to make a significant contribution.

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