The mitochondrial inner membrane is impermeable to NAD, and therefore the NADH produced in the cytosol in glycolysis cannot enter the mitochondria for reoxidation. In order to transfer the reducing equivalents from cytosolic NADH into the mitochondria, two substrate shuttles are used:
• The malate—aspartate shuttle (Figure 5.11) involves reduction of oxaloacetate in the cytosol to malate (with the oxidation of cytosolic NADH to NAD+). Malate enters the mitochondria and is reduced back to oxaloacetate, with the reduction of intramitochondrial NAD + to NADH. Oxaloacetate cannot cross the
mitochondrial inner membrane so undergoes transamination to aspartate (section 220.127.116.11), with glutamate acting as amino donor, yielding a-ketoglutarate. a-Ketoglutarate then leaves the mitochondria using an antiporter (section 3.2.2) which transports malate inwards. Aspartate leaves the mitochondria in exchange for glutamate entering; in the cytosol the reverse transamination reaction occurs, forming oxaloacetate (for reduction to malate) from aspartate, and glutamate (for transport back into the mitochondria) from a-ketoglutarate. The glycerophosphate shuttle (Figure 5.12) involves reduction of dihydroxyacetone phosphate to glycerol 3-phosphate in the cytosol (with oxidation of NADH to NAD+) and oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate inside the mitochondrion. Dihydroxyacetone phosphate and glycerol 3-phosphate are transported in opposite directions by an antiporter in the mitochondrial membrane.
The cytosolic glycerol 3-phosphate dehydrogenase uses NADH to reduce glycerol 3-phosphate dehydrogenase
cytosol o mitochondrion v ch2o-(p)
glycerol 3-phosphate dehydrogenase
Figure 5.12 The glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion.
dihydroxyacetone phosphate to glycerol 3-phosphate, but the mitochondrial enzyme uses FAD to reduce glycerol 3-phosphate to dihydroxyacetone phosphate. This means that when this shuttle is used there is a yield of ~2 X ATP rather than ~3 X ATP as would be expected from reoxidation of NADH.
The malate—aspartate shuttle is sensitive to the NADH/NAD+ ratios in the cytosol and mitochondria, and cannot operate if the mitochondrial NADH/NAD+ ratio is higher than that in the cytosol. However, because it does not use NAD+ in the mitochondrion, the glycerophosphate shuttle can operate even when the mitochondrial NADH/NAD + ratio is higher than that in the cytosol.
The glycerophosphate shuttle is important in muscle in which there is a very high rate of glycolysis (especially insect flight muscle); the malate—aspartate shuttle is especially important in heart and liver.
18.104.22.168 The reduction of pyruvate to lactate: anaerobic glycolysis
In red blood cells, which lack mitochondria, reoxidation of NADH formed in glycolysis cannot be by way of the substrate shuttles discussed above (section 22.214.171.124) and the electron transport chain.
Similarly, under conditions of maximum exertion, for example in sprinting, the rate at which oxygen can be taken up into the muscle is not great enough to allow for the reoxidation of all the NADH that is being formed in glycolysis. In order to maintain the oxidation of glucose, and the net yield of 2 X ATP per mol of glucose oxidized (or
3 mol of ATP if the source is muscle glycogen), NADH is oxidized to NAD+ by the reduction of pyruvate to lactate, catalysed by lactate dehydrogenase (Figure 5.13).
The resultant lactate is exported from the muscle and red blood cells and taken up by the liver, where it is used for the resynthesis of glucose. As shown on the right of Figure 5.13, synthesis of glucose from lactate is an ATP- (and GTP-) requiring process. The oxygen debt after strenuous physical activity is due to an increased rate of energy-yielding metabolism to provide the ATP and GTP that are required for gluconeogenesis from lactate. Although most of the lactate will be used for gluconeogenesis, a proportion will have to undergo oxidation to CO2 in order to provide the ATP and GTP required for gluconeogenesis (see Problem 5.1).
Lactate may also be taken up by other tissues in which oxygen availability is not a limiting factor, such as the heart. Here it is oxidized to pyruvate, and the resultant NADH is oxidized in the mitochondrial electron transport chain, yielding 3 X ATP The pyruvate is then a substrate for complete oxidation to carbon dioxide and water, as discussed below (section 5.4.3).
Many tumours have a poor blood supply and hence a low capacity for oxidative
metabolism, so that much of the energy-yielding metabolism in the tumour is anaerobic. Lactate produced by anaerobic glycolysis in tumours is exported to the liver for gluconeogenesis; as discussed in section 8.4, this increased cycling of glucose between anaerobic glycolysis in the tumour and gluconeogenesis in the liver may account for much of the weight loss (cachexia) that is seen in patients with advanced cancer.
Anaerobic glycolysis also occurs in micro-organisms that are capable of living in the absence of oxygen. Here there are two possible fates for the pyruvate formed from glucose, both of which involve the oxidation of NADH to NAD+:
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