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Download Chem*3560 Lecture 15: Gluconeogenesis
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Chem*3560 Lecture 15: Gluconeogenesis Gluconeogenesis is the synthesis of "new" glucose in the cytoplasm, and is undertaken when there is little demand for energy, and unused substrate is available. The liver is the major organ responsible responsible for gluconeogenesis in animals, and may take up lactate or amino acids from blood as a source of substrate (Lehninger p.723-729). The pathway may start in the cytoplasm from lactate, or in the mitochondrion use amino acids by removing the amino group. The gluconeogenesis pathway broadly follows glycolysis sequence in reverse. Eight reactions out of eleven are close to equilibrium (∆ ∆ G close to zero), and are therefore easily made to run in the opposite direction (‡ symbols in the diagram). Three reactions, hexokinase, phosphofructokinase, and pyruvate kinase have large negative ∆ G at cellular concentrations, and would be difficult or impossible to reverse. A different reaction or sequence is needed for each of these steps to proceed in the direction needed for gluconeogenesis. Conversion of pyruvate to phosphoenolpyruvate In animals, this is a multistep sequence. It is too difficult to add phosphate to pyruvate in a single reaction, because phosphoenolpyruvate (PEP) has an exceptionally high ∆ Go ' of hydrolysis (–61.9 kJ/mol). The strategy used is to add a carboxylate group to pyruvate first, which yields oxaloacetate. Since decarboxylation always releases considerable energy, an ATP must be used as an energy source when carboxylate is added. Then in a second reaction, the added carboxylate is lost again; with the help of GTP as a phosphate donor, the energy made available by this decarboxylation is used to drive the reaction in the direction of PEP formation. pyruvate carboxylase pyruvate + CO2 + ATP → oxaloacetate + ADP + Pi PEP carboxykinase oxaloacetate + GTP → PEP + CO2 + GDP Overall cost is 2 ATP equivalents to make PEP from pyruvate (Lehninger p. 726-727). Pyruvate carboxylase The enzyme, pyruvate carboxylase uses the coenzyme biotin to activate and localize the -CO2 – group. The enzyme has four identical subunits, with each polypeptide made up of three domains: Biotin carrier domain contains biotin covalently bonded to a lysine side chain. This forms a long "arm", and gives the biotin freedom of movement between the two catalytic sites. The biotin carboxylase domain uses ATP hydrolysis to drive addition of a bicarbonate ion HCO3 – to biotin (step 1), making carboxybiotin. The carboxyltransferase domain transfers the carboxylate group to the ultimate substrate, pyruvate, to yield oxaloacetate (step 2). This pattern of three components arranged around a bound biotin coenzyme is used by several other carboxylases, including acetyl CoA carboxylase (Lehninger p. 585-6). PEP carboxykinase PEP carboxykinase uses the decarboxylation of oxaloacetate to create the unstable enolpyruvate isomer of pyruvate, which can then accept phosphate from GTP. Location of pyruvate carboxylase and PEP carboxykinase Pyruvate carboxylase is located in mitochondria, whereas PEP carboxykinase is located in both cytoplasm and in mitochondria. Which is used depends on the starting substrate used. When lactate is the starting substrate, lactate dehydrogenase produces pyruvate and NADH in the cytoplasm. This cytoplasmic NADH is needed later to reduce 1,3-bisphosphophoglycerate. The cytoplasmic pyruvate must be imported through the mitochondrial membrane via a specific transporter, and is then converted to PEP in the mitochondria. PEP is exported by another transporter (Lehninger p. 728). When amino acids are used as starting substrate, the amino group is first removed and the carbon structures are converted to pyruvate, oxaloacetate or malate in mitochondria. Of these, only malate has an export transporter. The malate is then oxidized in the cytoplasm to provide the NADH that will be needed to reduce 1,3-bisphosphoglycerate. There is no transporter for NADH, so it must be generated in the cytoplasm when needed for gluconeogenesis. Once PEP is made and conditions favour gluconeogenesis, the enzymes of glycolysis can run in the opposite direction as far as fructose-1,6-bisphosphate. Fructose-1,6-bisphosphatase bypasses the phosphofructokinase step fructose-1,6-bisphosphatase fructose-1,6-bisphosphate + H2 O → fructose-6-phosphate + Pi ∆ Go ' = –15.9 kJ/mol This reaction is not the reverse of phosphofructokinase, because no ATP is produced. Both phosphofructokinase and fructose-bisphosphatase can have –ve ∆Go ', because they are different reactions (Lehninger p. 728). Glucose-6-phosphatase bypasses the hexokinase step Glucose-6-phosphate isomerase is freely reversible, but hexokinase represents the one-way reaction, which is bypassed by glucose-6-phosphatase glucose-6-phosphatase glucose-6-phosphate + H2 O → glucose + Pi ∆ Go ' = –13.8 kJ/mol Again, this is not the reverse of hexokinase because no ATP is produced (Lehninger p.729). Hydrolysis of Glucose-6-phosphate is coupled to export of glucose Most other enzymes of glycolysis and gluconeogenesis are located in the cytoplasm. Glucose-6-phosphatase is located in the cell membrane, and substrate is bound on the cytoplasmic side, but glucose product is released on the outside of the cell. This means that glucose is exported from the cell that makes it. Glucose-6-phosphatase is primarily an enzyme of the liver and kidneys, which routinely export glucose to maintain the blood glucose level. Muscles lack glucose-6-phosphatase, and direct glucose-6-phosphate to glycogen synthesis, keeping the glucose as a reserve within the cell than made it. Overall energy cost of gluconeogenesis is 4-6 ATP per glucose made Taking lactate as starting point, 2 lactates are needed per glucose 2 NADH made by lactate dehydrogenase 2 ATP needed by pyruvate carboxylase 2 GTP needed by PEP carboxykinase 2 ATP needed by phosphoglycerate kinase 2 NADH used glyceraldehyde-3-phosphate dehydrogenase No ATP made by fructose-1,6-bisphosphatase or glucose-6-phospahtase Net 6 ATP equivalents are needed to make one glucose (Lehninger p. 729). If the starting point is malate derived from amino acids, the pyruvate carboxylase step can be skipped, so that only 4 ATP are consumed.