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

Antiports ensure balanced transfers of key compounds

The ATP/ADP antiporter exchanges ATP4- for ADP3- at the mitochondrial membrane. The strict one in / one out stoichiometry balances input and output, ensuring no net change in total adenine nucleotide in the mitochondrion. There is a net charge change of -1 on the ATP4- destination side. Electron transport creates a membrane potential of 150-170 mM -ve inwards, so this favours outward movement of ATP4-. Each ATP transported will use up most of the energy of one H+ pumped out by electron transport.

Import of cytoplasmic NADH

There is no transporter for NADH produced in the cytoplasm, and instead the reducing equivalents of NADH are imported indirectly through shuttle mechanisms.

pathway for the reducing electrons

The glycerol phosphate shuttle is simpler, but less efficient and does not involve substrate transport: Cytoplasmic glycerol phosphate dehydrogenase reduces dihydroxyacetone-3-P to glycerol-3-P (thermodynamically favoured direction). Glycerol-3-P is reoxidised by a flavoprotein exposed on the outside face of the inner mitochondrial membrane. From FADH2, the electrons pass to ubiquinone and the electron transport chain (etc).

The glycerol-3-P has to pass through the mitochondrial outer membrane, but this is not a permeability barrier for molecules < 1 kDa. The reoxidation step involves FAD, so now the oxidation is thermodynamically favourable, but FADH₂ produces one less ATP than NADH in oxidative phosphorylation. This process is especially used in insect flight muscle where throughput is important.

The malate shuttle is a more efficient process, occuring in liver and cardiac muscle.

pathway for the reducing electrons	This process is based on reduction of cytoplasmic oxaloacetate by cytoplasmic NADH and import to the mitochondria of malate, and is the reverse of the process exporting oxaloacetate and reducing equivalents for gluconeogenesis. In the direction shown here, there is a problem: sustained operation of this sequence requires a cytoplasmic source of oxaloacetate.
	The enzyme transaminase (or aspartate aminotransferase) is found in both cytoplasmic and mitochondrial forms, and interconverts aspartate and oxaloacetate. This allows a cycle balancing the import and export of C4 compounds between mitochondria and cytoplasm The cycle can be sustained without net consumption of intermediates needed for other purposes.

However there is a problem with nitrogen balance and the question of antiports for malate and aspartate is not addressed yet.

pathway for the reducing electrons pathway for N-exchange

The complete malate shuttle system involves interconversion of glutamate and -ketoglutarate. -Ketoglutarate accepts the amino group from aspartate to make the corresponding C5 amino acid, glutamate; the enzyme transaminase uses the coenzyme pyridoxal-phosphate (PLP) as its internal N-acceptor. Glutamic acid exchanges for aspartate by antiport, and this cycle results in nitrogen balance being maintained between cytoplasm and mitochondria. The -ketoglutarate antiport exchanges malate for -ketoglutarate to maintain the balance of C5 compounds.

The system is totally balanced for maintining levels of N, C₄ and C₅ intermediates. However, the net direction needs to be addressed.

If both antiports are neutral, as represented in most textbooks, the direction would be governed by the reduction potentials of NAD⁺ in cytoplasmic and mitochondrial compartments. The ratio [NADH] / [NAD⁺] is typically 0.002 in the cytoplasm and 0.1 in the mitochondrion. NAD⁺ is a better oxidant and NADH is a poorer reductant in the cytoplasm, and this means that without some other driving force, the cycles will proceed in the opposite direction.

What makes the shuttle system proceed as desired is the electrogenic behavour of the Asp^- / H-Glu antiport (see LaNoue and Schoolwerth, Ann. Rev. Biochem., 48, 871-922 (1979)). The mitochondrial membrane potential drives negative aspartate outwards, but does not oppose entry of neutral glutamic acid. This provides the needed driving force to make the shuttle work in the right direction.

The role of the citrate antiport

The main regulatory step of the TCA cycle appears to be the energy dependent activity of isocitrate dehydrogenase (allosteric inhibition by ATP and product inhibition by NADH). When isocitrate dehydrogenase is minimally active, the unfavourable equilibrium of aconitase causes accumulation of large amounts of citrate rather than isocitrate.

H-Citrate2- exchanges for malate2- via the tricarboxylate antiporter, through which citrate may be delivered to the cytoplasm. The substrate requirement for H-citrate2- is essentially equivalent to symport of a proton with citrate3-, the normal ionization state at pH 7, so outward citrate flux is opposed by the mitochondrial proton gradient. Citrate is normally present in mitochondria at relatively high concentrations, so this has the effect of keeping citrate inside until its level is really high. Malate is a good antiport exchange substrate, since formation of citrate consumed an oxaloacetate. By returning a malate from the cytoplasm, the oxaloacetate is easily regenerated.

If necessary, the malate required for antiport exchange can be exported via the dicarboxylate antiport, which accepts HPO₄^2-. Oxidative phosphorylation creates a continuous demand for phosphate in mitochondria. However there can be other ways other ways to get cytoplasmic malate.

In many organisms, cytoplasmic citrate is a negative regulator of glycolysis. Citrate efflux from mitochondria occurs when ATP and NADH are high, therefore less glucose substrate needs to be committed to catabolism.

In liver and adipose tissue, citrate efflux to the cytoplasm starts the process of fatty acid biosynthesis. The presence of citrate lyase in the cytoplasm releases oxaloacetate and acetyl CoA. Acetyl CoA then enters the fatty acid biosynthesis pathway, while oxaloacetate is reduced back to malate for return to the mitochondrion.

The enzymes aconitase and isocitrate dehydrogenase also exist in a cytoplasmic form. Cytoplasmic isocitrate dehydrogenase is NADP+ dependent. Hence if mitochondria are satiated with NADH, substrate can be diverted out so as to make NADPH in the cytoplasm. The -ketoglutarate can reenter via its own antiporter. Malate is exchanged in for citrate coming out and is returned out in exchange for the re-entry of -ketoglutarate, so this cycle produces no net change in malate pools.

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