Gluconeogenesis phosphoenolpyruvate production

Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are present primarily in the cytoplasm. Oxaloacetate, the product of the pyruvate carboxylase reaction, must thus be transported to the cytoplasm to complete the pathway. Oxaloacetate is transported from a mitochondrion in the form of malate oxaloacetate is reduced to malate inside the mitochondrion by an NADH-linked malate dehydrogenase. After malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD -linked malate dehydrogenase in the cytoplasm (Figure 16.26). The formation of oxaloacetate from malate also provides NADH for use in subsequent steps in gluconeogenesis. Finally, oxaloacetate is simultaneously decarboxylated and phospho-ry lated by phosphoenolpyruvate carboxy kinase to generate phosphoenol pyruvate. The phosphoryl donor is GTP. The GO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. 

Comparison of the reactions of glycolysis and gluconeogenesis. All the reactions of glycolysis occur in the cytoplasm of the cell. However, in many human cells, pyruvate carboxylase is found in the mitochondria and phosphoenolpyruvate carboxykinase is located in the cytoplasm. Oxaloacetate, the product of the reaction catalyzed by pyruvate carboxylase, is shuttled out of the mitochondria and into the cytoplasm by a complex set of reactions. 

Insulin inhibits transcription of the enzyme phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is a key enzyme in gluconeogenesis and transcription is the primary means of regulating it. By inhibiting PEPCK transcription, insulin can depress glucose production tremendously. (Conversely, the hormone glucagon, which increases blood glucose levels, stimulates PEPCK transcription.)... 

Fig 8. Production of blood glucose from glycogen (by glycogenolysis) and from alanine, lactate, and glycerol (by gluconeogenesis). PEP = phosphoenolpyruvate OAA = oxaloacetate. 

Fig. 31.5. Conversion of pyruvate to phosphoenolpyruvate (PEP). Follow the shaded circled numbers on the diagram, starting with the precursors alanine and lactate. The first step is the conversion of alanine and lactate to pyruvate. Pyruvate then enters the mitochondria and is converted to OAA (circle 2) by pyruvate carboxylase. Pyruvate dehydrogenase has been inactivated by both the NADH and acetyl-CoA generated from fatty acid oxidation, which allows oxaloacetate production for gluconeogenesis. The oxaloacetate formed in the mitochondria is converted to either malate or aspartate to enter the cytoplasm via the malate/aspartate shuttle. Once in the cytoplasm the malate or aspartate is converted back into oxaloacetate (circle 3), and phosphoenolpyruvate carboxykinase will convert it to PEP (circle 4). The white circled numbers are alternate routes for exit of carbon from the mitochondrion using the malate/aspartate shuttle. OAA = oxaloacetate FA = fatty acid TG = triacylglycerol.

The equilibrium of pyruvate kinase is also strongly in the direction of glycolysis, because the immediate product of the reaction is enolpyruvate, which is chemically unstable. As shown in Figure 5.31, enolpyruvate undergoes a non-enzymic reaction to yield pyruvate. This means that little of the product of the enzymic reaction is available to undergo the reverse reaction in the direction of gluconeogenesis. The conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis is discussed in section 5.7. 

Qtrate, which can also be considered to be an end-product of glycolysis, also inhibits phosphofructokinase, by enhancing the inhibition by ATP. In muscle, creatine phosphate (section 3.2.3.1) has a similar effect. Phosphoenolpyruvate, which is synthesized in increased amounts for gluconeogenesis (section 5.7), also inhibits phosphofructokinase. 

Cocoa bean fermentation is a mixed-culture process, consisting initially of fermentations by yeast and lactic acid bacteria followed by oxidation of the fermentation products ethanol and lactic acid into acetic acid and acetoin by several Acetohacter strains, of which /I. pasteurianus is the prominent one (Moens et al. 2014). A C-based carbon flux analysis of Acetohacter during cocoa pulp fermentation-simulating conditions revealed a functionally separated metabolism during co-consumption of ethanol and lactate. Acetate was almost exclusively derived from ethanol, whereas lactate served for formation of acetoin and biomass building blocks. This switch was attributed to the lack of phosphoenolpyruvate carboxykinase and malic enzyme activities, which prevents conversion of oxalo-acetate and malate formed by acetate metabolism in the TCA cycle to PEP and pyruvate and subsequently to acetoin (Adler et al. 2014). Lactate, on the other hand, can be converted to pyruvate, which is then used for acetoin formation or, after conversion to PEP by pymvate phosphate dikinase, for gluconeogenesis. The inability of conversion of TCA cycle intermediates to PEP resembles the situation in G. oxydans, where in addition no enzyme for conversion of pyruvate to PEP is present. 

---