III. Metabolism - Gluconeogenesis

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1 Department of Chemistry and Biochemistry University of Lethbridge III. Metabolism - Gluconeogenesis Carl & Gertrude Cori Slide 1

2 Carbohydrate Synthesis Lactate, pyruvate and glycerol are the important 3C compounds that feed gluconeogenesis. Glucogenic amino acids are catabolized to pyruvate, and other citric acid cycle compounds that can enter gluconeogenesis. Photosynthetic organisms use the pathway to fix CO2. Occurs in the liver of mammals! Slide 2

3 Glucogenic Amino Acids? Slide 3

4 The Cori Cycle When a working muscle goes anaerobic, the generated lactate is excreted, then transported to the liver via the bloodstream. Liver: lactate is converted to glucose (gluconeogenesis). Glucose (liver) is made available, through the bloodstream, to other tissues e.g. muscle. Resting muscle: Glucose is still transported to the muscle to replenish glycogen levels. Slide 4

5 Glycolysis Again Slide 5

6 Gluconeogenesis Glycolysis and gluconeogenesis share 7 of 10 steps Reactions of hexokinase, PFK-1 and pyruvate kinase are essentially irreversible in vivo. These steps are bypassed in gluconeogenesis and require new, different enzymes. The gluconeogenesis enzyme reactions are also irreversible in vivo. Both are reciprocally regulated, cytosolic pathways. Metabolite flux is always in one direction. Slide 6

7 Gluconeogenesis Reactions Glucose 6-phosphatase and Fructose 1,6- bisphosphatase catalyze simple hydrolysis reactions. strongly favorable Slide 7

8 Gluconeogenesis Reactions Conversion of pyruvate to PEP is complex. Pyruvate is transported into the mitochondrion to enter gluconeogenesis Oxaloacetate is both a citric-acid cycle and a gluconeogenesis metabolite occurs in mitochondria Slide 8

9 Gluconeogenesis Step 1 Two different pathways are possible! Difference: malate dehydrogenase Regulation: depends on the presence of lactate in the cytosol. The mitochondrion has no oxaloacetate transporter! Note: pyruvate carboxylase requires ATP hydrolysis & PEP carboxykinase requires GTP hydrolysis Slide 9

10 Gluconeogenesis Step 1 Pyruvate carboxylase Biotin aka vitamin B7 or vitamin H (another coenzyme) 1 ATP is used! C-C bond formed Slide 10

11 Gluconeogenesis Step 1 Biotin is covalently attached to the the ε-amino group of lysine. Reaction involves two different active sites on the same enzyme. Site 1: Form carboxybiotin from HCO3- and ATP Site 2: Form oxaloacetate from pyruvate and released CO2 Slide 11

12 Gluconeogenesis Step 1 Active site 1 Bicarbonate is converted to CO2 which reacts with Biotin forming carboxybiotin Active site 2 Carboxybiotin releases CO2 which reacts with pyruvate forming oxaloacetate Slide 12

13 Gluconeogenesis Step 2 PEP carboxykinase Oxaloacetate is converted to PEP using GTP as phosphoryl donor. GTP is often used as a energy source in anabolism Slide 13

14 Gluconeogenesis Steps 1 & 2 Net cost for the reactions is 2 ATP (or 1 ATP + 1 GTP) PEP carboxykinase reversibly exchanges high energy bonds (GTP for PEP) G o = 0.9 kj/mol, but under cellular conditions G = -25 kj/mol Slide 14

15 Summary Slide 15

16 Pentose Phosphate Pathway Alternative path for glucose oxidization Electron acceptor is NADP+. Products: pentose phosphates + NADPH Pentose phosphates NADPH is needed for reductive biosynthesis AND prevents oxidative damage to proteins at high levels (red blood cells, cornea) Slide 16

17 Pentose Phosphate Pathway Oxidative Phase cyclic ester hydrolysis oxidative decarboxylation isomerization Oxidations (dehydrogenases) have large negative free energy changes and are essentially irreversible. Two NADPH are produced per G6P starting molecule - by the dehydrogenase reactions. 1 pentose phosphates is produced per G6P starting molecule Slide 17

18 Pentose Phosphate Pathway Oxidative Phase Step 1: Cyclic ester product Cyclic aldose sugar to cyclic sugar acid Slide 18

19 Pentose Phosphate Pathway Oxidative Phase Step 2: Hydrolysis of cyclic ester Cyclic sugar acid to linear sugar acid Slide 19

20 Pentose Phosphate Pathway Oxidative Phase Step 3: Oxidative decarboxylation Acid hexose to ketopentose Slide 20

21 Pentose Phosphate Pathway Oxidative Phase Step 4: Isomerisation Ketopentose to aldopentose Slide 21

22 Pentose Phosphate Pathway Nonoxidative Phase?? Choreographed Dance?? Ribulose-5-phosphate is converted back into Glucose-6-phosphate Slide 22

23 Pentose Phosphate Pathway Nonoxidative Phase Overall: 5C sugars are converted to 6C sugars. G6P is regenerated from pentose phosphates to make more NADPH. Note: Epimerase actually converts ribulose 5-phosphate to xylulose 5-phosphate PP pathway shares intermediates with glycolysis/gluconeogenesis Enzymes are all cytosolic and unique to PP pathway Slide 23

24 Pentose Phosphate Pathway - Nonoxidative Reactions 5 hexoses (6C) are made from 6 pentoses (5C) Every reaction shown here is reversible! Hexoses (blue) are fructose-6-phosphate Pentoses (pink) are derived from ribulose-5-phosphate Slide 24

25 Epimerase Ribulose 5-phosphate 3-epimerase Reaction utilizes an enediol intermediate similar to phosphopentose isomerase. Epimerase reaction abstracts then adds a proton to C3, results in an inversed configuration on the carbon atom. Slide 25

26 Transketolase Transfer of 2-carbon group TPP-mediated Remember the Coenzyme? - still bond-breaking but substrate is not an α-keto acid Slide 26

27 Thiamine Deficiency The ability of TTP s thiazolium ring to add carbonyl groups and act as an electron sink makes it the coenzyme most utilized in α-keto acid decarboxylations. Thiamin (vitamin B1) is neither synthesized nor stored in significant amounts by vertebrates. Deficiency in humans results in an ultimately fatal condition known as beriberi. Slide 27

28 Transaldolase Transfer of 3-carbon unit; similarity to aldolase cleavage reaction in glycolysis. Slide 28

29 Short Reminder Class I Aldolase Schiff's base mediated C-C bond cleavage Slide 29

30 Mechanistic Similarity! Transketolase TPP stabilizes the two-carbon carbanion Transaldolase Schiff's base stabilizes the three-carbon carbanion Slide 30

31 Regulation Excess NADPH Glucose 6-phosphate dehydrogenase is allosterically inhibited by NADPH. ie. Feedback inhibition of committed step of pentose phosphate pathway Slide 31