Gluconeogenesis

• gluconeogenesis = conversion of non-carbohydrate precursors into glucose
• gluconeogenesis is not exactly the reverse of glycolysis, because pyruvate must go through the oxaloacetate intermediate before making glucose
• gluconeogenesis can form glucose from molecules such as pyruvate, lactate, glycerol, amino acids, TCA intermediates
• gluconeogenesis requires different enzymes than glycolysis at the irreversible steps of glycolysis
• when converting acetyl-CoA to glucose, there is no net increase in carbon, oxaloacetate, and ultimately glucose
• gluconeogenesis consumes 4 ATP and 2 GTP
• gluconeogenesis mostly occurs in the liver
• why there are different pathways for glycolysis and gluconeogenesis:

  1. need to make both directions thermodynamically favorable (think of the irreversible steps covered in glycolysis)
  2. regulation purposes -- can specify which pathway will occur and specialize where they occur, can regulate reciprocally
• oxaloacetate is a higher energy intermediate formed in gluconeogenesis
• pyruvate carboxylase, which catalyzes the formation of oxaloacetate for gluconeogenesis, requires the cofactor biotin (vitamin B7)
• biotin ligase catalyzes the covalent attachment of biotin to a lysine residue of pyruvate carboxylase
• the attachment of biotin to pyruvate carboxylase (PC) occurs in two steps:

  1. adenylation, which requires ATP, to activate biotin's carboxylate group
  2. attachment to PC through lysine
• adenylation = uses ATP for the covalent addition of AMP; compared to phosphorylation, it provides more energy, and the first phosphate is attacked instead of the terminal phosphate on ATP
• pyruvate carboxylase has three domains (in order from N to C terminus):

  1. biotin carboxylase (BC) domain
  2. carboxyl transferase (CT) domain
  3. biotin carboxyl carrier protein (BCCP) domain, which contains the lysine that biotin gets attached to
• three steps of pyruvate carboxylase to form oxaloacetate from pyruvate:

  1. bicarbonate (soluble CO2) is phosphorylated by ATP to form carboxyphosphate
  2. CO2 is transferred from carboxyphosphate to biotin
  3. CO2 is transferred from biotin to pyruvate; Zn2+ dependent step to stabilize the negative charges of deprotonated pyruvate
• biotin is required for pyruvate carboxylase, where it ultimately acts as a CO2 carrier on the end of a long arm to deliver it to the appropriate active site
• role of three domains of pyruvate carboxylase:

  1. BC active site: where bicarbonate is phosphorylated and CO2 from it is transferred to biotin
  2. CT active site: transfers CO2 to pyruvate from biotin
  3. BCCP: acts as the biotin carrier, a long arm that can swing between BC and CT to move biotin to proper locations
• Quaternary structure of pyruvate carboxylase? how does this help the enzyme function?

  Pyruvate carboxylase is a tetramer! the CT domain and BC domain of opposite monomers are actually closest to each other, and so CO2 is actually shuttled between domains of different monomers by BCCP domain

  
• Carboxylate pyruvate with pyruvate carboxylase (PC) and decarboxylate it again with PEPCK
• Without the oxaloacetate intermediate, phosphorylating pyruvate to PEP is thermodynamically unfavorable
• Carboxylation of pyruvate forms oxaloacetate, a higher-energy intermediate
• Favorable decarboxylation drives an otherwise uphill, unfavorable reaction
• Why carboxylate pyruvate, just to decarboxylate it again?

  Without the oxaloacetate intermediate, phosphorylating pyruvate to PEP is thermodynamically unfavorable. Favorable decarboxylation drives an otherwise uphill, unfavorable reaction. Energy from decarboxylation can make up for the ATP needed for the first carboxylation step, resulting in a net delta G of about 0
• The first two steps of glycolysis uses ATP to drive otherwise unfavorable phosphorylation reactions. So then in gluconeogenesis, we can just do the reverse favorable reaction, hydrolyzing off phosphate
• Regulation of glycolysis and gluconeogenesis is reciprocal
• Mitochondrial gluconeogenesis steps:

  • pyruvate carboxylase (forming oxaloacetate intermediate)
  • and sometimes PEPCK (decarboxylation)
• Cytosolic PEPCK is used when pyruvate is directly used for gluconeogenesis. Mitochondrial PEPCK is used when lactate is used. Overall, the goal is to increase cytoplasmic levels of NADH.
• When cytosolic PEPCK is used, the oxaloacetate will be converted to malate in the mitochondria, consuming NADH, and it is the malate that is brought of the mitochondria to the cytoplasm. There, malate is converted back to oxaloacetate, producing NADH in the cytoplasm, and then cytosolic PEPCK can act on the cytoplasmic oxaloacetate to form PEP.
• When mitochondrial PEPCK is used, oxaloacetate can be converted to PEP directly in the mitochondria after the pyruvate carboxylase step. The goal is to increase cytoplasmic levels of NADH, and when mitochondrial PEPCK is used, this is already achieved because the conversion of lactate to pyruvate in the cytoplasm (before the pyruvate enters the mitochondria for gluconeogenesis PC and PEPCK steps) already generates NADH in the cytoplasm.
• Why do we use cytosolic or mitochondrial PEPCK accordingly in order to increase cytoplasmic levels of NADH? (Basically, why do we want to increase cytoplasmic levels of NADH?)

  We need NADH for a later step of gluconeogenesis, which occurs only in the cytoplasm after this point! Basically, we are preparing for later parts of gluconeogenesis
• In the lactate or Cori cycle, lactate and glucose are circulated between muscle and liver cells, where gluconeogenesis in the liver provides glucose for glycolysis in muscles, which in turn provides lactate through fermentation for the liver.