Biochemistry - I SPRING Mondays and Wednesdays 9:30-10:45 AM (MR-1307) Lecture 16. Based on Profs. Kevin Gardner & Reza Khayat

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1 Biochemistry - I Mondays and Wednesdays 9:30-10:45 AM (MR-1307) SPRING 2017 Lecture 16 Based on Profs. Kevin Gardner & Reza Khayat 1

2 Catabolism of Di- and Polysaccharides Catabolism (digestion) begins in the mouth where salivary α-amylase (digest α1 4) hydrolyzes the glycosidic linkages of starch (physiological ph ~6.8) Salivary α-amylase inactive in low ph of stomach, but pancreatic α-amylase secreted into small intestine is active and continues the digestion to produce maltose and maltotriose (di- and trisaccharides of glucose). These are converted to D-glucose by maltase. Also remaining are the branched saccharides (α1 6). 2

3 Dextrin Maltose (α,α,1 4) Glucose Sucrose (α,β,1 2) Trehalose (α,α,1 1) 3

4 Catabolism of Galactose Enters glycolysis after several reactions Galactose phosphorylated on C-1 Galactose 1-phosphate takes the uridine diphosphate (UDP) sugar-nucleotide from UDP-glucose (a product of this reaction) to generate glucose 1-phosphate for glycolysis and UDP-galactose UDP-galactose is oxidized on C-4 to a ketone It is then reduced stereospecifically at C-4 to its enantiomer (epimer) UDP-glucose UDP-glucose is used in step 2 UDP acts as a coenzyme carrier of hexoses (helps with catalysis) sugar UDP sugar TYPO!! SHOULD BE REVERSE 4

5 Catabolism of Glycogen Primarily in skeletal muscle and hepatocytes (liver) Glycogen phosphorylase catalyzes attack by inorganic phosphate on the terminal glucosyl residue at nonreducing end of a glycogen (Ch. 15) Glucose 1-phosphate is released for glycolysis (G1P) G1P converted to G6P by phosphoglucomutase, and into glycolysis 5

6 Fates of Pyruvate Post-glycolysis 6

7 Fermentation: Fate of Pyruvate under Anaerobic Conditions Extensive exercise, submerged plant tissues, solid tumors, erythrocytes (red blood cells lack organelles) have low levels of O2. Without O2, NADH generated during glycolysis can not be oxidized to NAD + by mitochondria. Oxidation of NADH by mitochondria via the electron transport chain produces additional ATP Pyruvate becomes the oxidizing agent for NADH oxidation. Under this condition, there is no net gain of NAD + and generation of ATP by electron transport chain 7

8 Microbial Fermentation Yeast and other microorganisms ferment glucose to ethanol and CO2, rather than lactate Product of glycolysis, pyruvate, is decarboxylated and reduced Thiamine pyrophosphate (TPP) O C C O O CH 3 Pyruvate TPP, Mg 2 CO 2 pyruvate decarboxylase O C H CH 3 Acetaldehyde NADH H NAD alcohol dehydrogenase OH CH 2 CH 3 Ethanol Glucose 2ADP 2P i 2 ethanol 2CO 2 2ATP 2H 2 O Released CO2 is responsible for the bubbles in beer, champagne and dough rising Alcohol dehydrogenase runs the opposite reaction = production of acetaldehyde. Acetaldehyde binds to proteins to form adducts that are linked to organ disease and cancer 8

9 Summary Glycolysis: glucose ATP + NADH equivalents and carbon left as pyruvate Most sugars enter glycolysis as glucose or fructose Pyruvate enters TCA cycle to turn into CO2 (aerobic) or ferments to lactic acid (anaerobic); EtOH in microbes Next: Can we run glycolysis backwards to make glucose? Can glycolytic intermediates be utilized for other purposes in the cell? 9

10 Gluco neo genesis Conversion of pyruvate and related three- or four-carbon compounds to glucose Occurs when glucose levels are really low and there is not enough glycogen in muscle and liver to supply it (e.g. during fasting, vigorous exercise, long tests...) Steps almost the reverse of glycolysis; must bypass reactions that are nearly irreversible in the cell (reactions 1, 3, 10) Bypasses are also irreversible, thus glycolysis and gluconeogenesis are both nearly irreversible Note that the differences in steps mean that the chemical balance of glycolysis is not reverse of gluconeogenesis A fraction of enzymes conduct glycolysis while other fraction conducts gluconeogenesis Takes place in the liver, renal cortex (portion of kidney), and epithelial cells that line the inside of small intestine Glucose passes into blood and carried to needed tissues Energetically expensive, but essential given that the brain uses 120 g of glucose a day 10

11 Why isn t Gluconeogenesis the Reverse of Glycolysis? R1 R3 G RT ln K n. eq aa bb cc dd G G RT ln [C]c [D] d [A] a [B] b terms in red are those actuall Glycolysis: red arrows, top to bottom Gluconeogenesis: blue arrows, bottom to top R10 11

12 Gluconeogenesis Bypass 1 (=Glycolysis R10) First bypass is the synthesis of phosphoenolpyruvate from pyruvate Pyruvate is converted to oxaloacetate in mitochondria Oxaloacetate is converted to phosphoenolpyruvate in the cytosol from CO2 Pyruvate ATP GTP HCO3 PEP ADP GDP P i CO 2 (14 8) G 0.9 kj/mol Pyruvate carboxylase is first regulatory enzyme in gluconeogenesis. It requires acetyl- CoA as a positive effector (produced by FA oxidation). Lots of acetyl-coa means lots of energy present from FA oxidation thus turn on gluconeogenesis. (PEP) Conversion of PEP to pyruvate by pyruvate kinase (glycolysis R10) is inhibited by phosphorylation of pyruvate kinase (turns off glycolysis). 12

13 Gluconeogenesis Bypass 1 (=Glycolysis R10) Two competing pathways initiate gluconeogenesis, differing in NADH generation strategies [lactate] determines pathway 1 or 2 Enzyme to make oxaloacetate is only in mitochondrion Cytosolic [NADH] is low and needs to be replenished Mitochondrial [NADH] is high cytosolic PEP carboxykinase Oxaloacetate CO 2 PEP 1. Cytosolic [NADH] replenished via malate shuttling 2. Cytosolic [NADH] replenished via oxidation of lactate to pyruvate cytosolic malate dehydrogenase Malate NADH + H + NAD + Regardless of 1 vs. 2, PEP continues with gluconeogenesis Remember - bacteria do not have mitochondria Malate PEP mitochondrial malate dehydrogenase Oxaloacetate NAD + NADH + H + mitochondrial PEP carboxykinase CO 2 Oxaloacetate pyruvate carboxylase CO 2 pyruvate carboxylase CO 2 Pyruvate Mitochondrion Pyruvate Cytosol Pyruvate 1 lactate dehydrogenase Pyruvate Lactate 2 NADH + H + NAD + 13

14 Gluconeogenesis Bypass 2 (=Glycolysis R3) Second bypass is the dephosphorylation (hydrolysis) of fructose 1,6-bisphosphate to fructose 6-phosphate. Note the generation of Pi Catalyzed by fructose 1,6-bisphosphatase (FBPase-1). FBPase-1 is regulated via phosphorylation by a kinase. Fructose 1,6-bisphosphate H 2 O fructose 6-phosphate P i G 16.3 kj/mol 14

15 Gluconeogenesis Bypass 3 (=Glycolysis R1) Third bypass is conversion of glucose 6-phosphate to glucose As with bypass 2, this is a dephosphorylation step, this time catalyzed by glucose 6-phosphatase. Note the generation of Pi Enzyme only present in hepatocytes, renal cells and epithelial cells of small intestine. Thus gluconeogenesis is only possible in these cells! Glucose 6-phosphate H 2 O glucose P i G 13.8 kj/mol 15

16 Summary of Gluconeogenesis For each molecule of glucose formed from pyruvate, six high energy phosphate groups are required, four from ATP and two from GTP Two molecules of NADH for the reduction of two molecules of 1,3-bisphosphoglycerate Note the summation: gluconeogenesis glycolysis Glucose 2ATP 2NAD 4ADP 2P i 88n 2 pyruvate 2ADP 2NADH 2H 4ATP 2H 2 O Glycolysis and gluconeogenesis are reciprocally regulated in cells that can do both 16

17 Amino Acids as a Source of Pyruvate Almost all amino acids can be converted to pyruvate through an intermediate of the citric acid cycle (TCA), allowing conversion of protein -> AA -> glucose Oxaloacetate is an intermediate of TCA Useable AAs are called glucogenic Note that Leu and Lys are not glucogenic 17

18 Summary III Glycolysis: glucose ATP + NADH equivalents and carbon left as pyruvate Most sugars enter glycolysis as glucose or fructose Pyruvate enters TCA cycle to turn into CO2 (aerobic) or ferments to lactic acid (anaerobic); EtOH in microbes Gluconeogenesis allows the synthesis of glucose from pyruvate in a pathway using many, but not all, steps of glycolysis Next: can glycolytic intermediates be utilized for other 18

19 Pentose Phosphate Pathway Needed by rapidly dividing cells (bone marrow, skin, intestinal mucosa); need pentoses to make DNA, RNA, ATP, NADPH, FADH 2, and coenzyme A Needed by tissues exposed directly to oxygen (RBCs, lens, cornea) because they have lots damaging free radicals. Reducing atmosphere (high ratio of NADPH to NADP +, and high ratio of reduced to oxidized glutathione) minimizes oxidative damage NADPH also needed for biosynthesis 19

20 Pentose Phosphate Pathway Oxidized! Reduced! End products are ribose 5-P, CO 2, and NADPH The net result is the production of NADPH, a reductant, and ribose 5-P, a precursor for nucleotide biosynthesis Oxidized! Reduced! Glucose 6-phosphate 2NADP H 2 O ribose 5-phosphate CO 2 2NADPH 2H The net result is the production of NADPH, a reductant 20

21 [NADPH] Regulates G-6-P Fate Glucose 6-P can enter glycolysis or the pentose phosphate pathway When NADPH is forming faster than it is being used for biosynthesis and glutathione reduction, NADPH concentration rises and it inhibits the first enzyme in the pentose phosphate pathway (glucose 6-P dehydrogenase; below) 21

22 Glucose 6-phosphate Dehydrogenase Glutathione (gamma-glu /Cys /Gly) is a major redox controller of cell Reduced glutathione (GSH) protects cells by destroying H2O2 and OH Regeneration of GSH from its oxidized form (GSSG) requires the NADPH produced in the glucose 6-P dehydrogenase reaction glutathione Deficiency of G6-P dehydrogenase under oxidizing conditions will lead to oxidative damage G6-P dehydrogenase deficiency is frequent where malaria is common because malaria parasite(s) living in erythrocytes are sensitive to oxidative damage. The G6-P dehydrogenase deficiency exists because it is tolerable to the human host and thus gives them an advantage 22