Gluconeogenesis Synthesis of "new glucose" from common metabolites Humans use ~160 g of glucose per day 75% is used by the brain Body fluids contain only 20 g of glucose Glycogen stores yield 180-200 g of glucose So the body must be able to make its own glucose 90% of gluconeogenesis occurs in the liver and kidneys

Figure 18.1 The Glycolysis Pathway

Figure 18.1 The Glycolysis Pathway

Why is gluconeogenesis not just the reverse of glycolysis? The reverse of glycolysis is 2 Pyruvate + 2ATP + 2 NADH + 2H+ + 2H20 a glucose +2ADP +2Pi + 2 NAD + (DG = +74 kJ/mol) This is thermodynamically unfavorable, so energetically unfavorable steps in the reverse glyolysis reaction are replaced and energy is added in the form of GTP and ATP to give: The actual equation for gluconeogenesis of 2Pyruvate + 4ATP +2GTP+ 2NADH + 2H+ + 6H20 a glucose +4ADP +2GDP +6Pi + 2 NAD + (DG = -38 kJ/mol) Notice the extra ATPs and GTPs drive the process

Glycolysis vs Gluconeogenesis Glycolosis Glucose (6C) to 2 pyruvates (3C) Creates energy 2ATP Reduces 2 NAD+ to 2 NADH Active when energy in cell low 10 steps from glucose to pyruvate Pyruvate to AcCoA before Krebs Gluconeogenesis 2 pyruvates (3C) to Glucose (6C) Consumes energy 4ATP+2GTP Oxidizes 2NADH to 2 NAD+ Active when energy in cell high 11 steps from pyruvate to glucose AcCoA isn’t used in gluconeogenesis Gluconeogenesis uses 7 of the 10 enzymatic reactions of glycolysis but in the reverse direction. The 3 not used are the ones requiring ATP in glycolysis.

FIGURE 22. 1 The pathways of gluconeogenesis and glycolysis FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate).

The pyruvate carboxylase reaction. First Reaction of Gluconeogenesis - recall that pyruvate is the final product of glycolysis. The pyruvate carboxylase reaction. (Simplified)

Biotin is an essential cofactor in most carboxylation reactions. It is an essential vitamin in the human diet, but deficiencies are rare. Avidin, a protein found in egg white binds tightly to biotin and excessive consumption of raw egg white can lead to biotin deficiency. FIGURE 22.3 Covalent linkage of biotin to an activesite lysine in pyruvate carboxylase.

ATP oxaloacetate Carbonyl phosphate FIGURE 22.4 A mechanism for the pyruvate carboxylase reaction. Bicarbonate must be activated for attack by the pyruvate carbanion. This activation is driven by ATP and involves formation of a carbonylphosphate intermediate—a mixed anhydride of carbonic and phosphoric acids. (Carbonylphosphate and carboxyphosphate are synonyms.) oxaloacetate

Pyruvate is converted to oxaloacetate in the mitochondria Oxaloacetate cannot be transported directly across the mitochondrial membrane so it is converted to malate, then transported, then oxidized back to oxaloacetate.

The PEP carboxykinase reaction.

Nucleotide diphosphate kinases Both glycolysis and Oxidative phosphorylation produce ATP with its high energy phoshoanhydride bonds: How does GTP get made from GDP? Directly from a single step in the Krebs cycle AND from the following reaction GDP + ATP → GTP + ADP This is carried out in the cell by an enzyme called Nucleotide diphosphate kinase which carries out the general reaction NDP + ATP → NTP + ADP (where N is T, G, or C)

FIGURE 22. 1 The pathways of gluconeogenesis and glycolysis FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate). 14

Enolase Reaction glycolysis gluconeogenesis Fig. 18-26, p. 595 FIGURE 18.26 The enolase reaction. Fig. 18-26, p. 595

FIGURE 22. 1 The pathways of gluconeogenesis and glycolysis FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate). 16

The Phosphoglycerate Mutase Reaction glycolysis gluconeogenesis FIGURE 18.23 The phosphoglycerate mutase reaction. Fig. 18-23, p. 594

Isomerase: An enzyme that catalyzes the transformation of compounds into their positional isomers. In the case of sugars this usually involves the interconversion of an aldose into a ketose, or vice versa. Kinase: An enzyme that catalyzes the phosphorylation (or dephosphorylation) of a molecule using ATP (or ADP). Mutase: An enzyme that catalyzes the transposition of functional groups, such as phosphates, sulfates, etc.

FIGURE 22. 1 The pathways of gluconeogenesis and glycolysis FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate). 19

Phospoglycerate kinase glycolysis gluconeogenesis FIGURE 18.20 The phosphoglycerate kinase reaction. Phospoglycerate kinase Fig. 18-20, p. 593

FIGURE 22. 1 The pathways of gluconeogenesis and glycolysis FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate). 21

The glyceraldehyde-3-phosphate dehydrogenase reaction glycolysis gluconeogenesis FIGURE 18.17 The glyceraldehyde-3-phosphate dehydrogenase reaction.

FIGURE 22. 1 The pathways of gluconeogenesis and glycolysis FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate). 23

Triose phosphate isomerase glycolysis gluconeogenesis FIGURE 18.14 The triose phosphate isomerase reaction. Triose phosphate isomerase Fig. 18-14, p. 589

FIGURE 22. 1 The pathways of gluconeogenesis and glycolysis FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate). 25

Aldolase 4th reaction of glycolysis (7th reaction of gluconeogenesis). Reversible reaction also used in gluconeogenesis. An aldol cleavage reaction (the reverse of an aldol condensation). glycolysis gluconeogenesis

FIGURE 22. 1 The pathways of gluconeogenesis and glycolysis FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate). 28

ANIMATED FIGURE 18.4 Phosphorylation of glucose to glucose- 6-phosphate by ATP creates a charged molecule that cannot easily cross the plasma membrane. See this figure animated at http://chemistry. brookscole.com/ggb3 Fig. 18-4, p. 584

Glucose-6-phosphatase enzyme unique to liver and kidney allowing them to supply glucose to other tissues. Found in ER

The Cori Cycle

Regulation of Gluconeogenesis Glucose-6-phosphatase is subject to substrate level control. - at higher G6P concentrations reaction rate increases - recall, this happens in the liver. Other tissues do not hydrolyze their G6P, thereby trapping it in the cells. Glycolysis and gluconeogenesis are reciprocally regulated. - regulatory molecules that inhibit gluconeogenesis often activate glycolysis, and vise versa.

A potent allosteric regulatory molecule. - activates phosphofructokinase. - inhibits fructose-1,6-bisphosphatase. - its synthesis and degradation are catalyzed by the same bifunctional enzyme.

Fructose-2,6-bisphosphate activates glycolysis and inhibits gluconeogenesis, so its level is very important.

F2,6 BP PFK-1 F2,6 BP ATP ADP Pi PFK-2 F2,6 BP INHIBITS STIMULATES FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate). 37

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Increased phosphorylation 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase Low glucose High glucagon Increased phosphorylation Phosphorylation of the enzyme results in the inactivation of the phosphofructokinase-2 activity and activation of the fructose-2,6-bisphosphatase activity. This results in a down regulation of glycolysis and increased gluconeogenesis.

Substrates for gluconeogenesis: Pyruvate Lactate TCA cycle intermediates Most amino acids Not substrates for gluconeogenesis: Acetyl-CoA Fatty acids Lysine Leucine

Plants and bacteria can make glucose from acetate.