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2010-11-11 11:49:09
Biomolecular Science

Metabolism- Glycolysis
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  1. What shape is the Glycolysis pathway?
  2. Glycolysis (general facts)
    • series of biochemical rxns that convers 1 glucose molecule to 2 molecules of pyruvate with release of usable E in form of 2 ATP molecules
    • an anaerobic process in the cytoplasm
    • on it's own an inefficient metabolic pathway for E generation
    • pyruvate can be futher fermented to lactate or ethanol
  3. one molecule of glucose = ?
    one molecules of glucose = (2) 3-C molecules (of pyruvate) and 2 molecules of ATP
  4. Major stages in glycolysis
    • I- conversion of glucose to fructose-1,6-bisphosphate (2 ATP used)
    • II- generation of 2 inter-convertible 3-carbon molecules, dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)
    • III- conversion of DHAP and GAP to pyruvate (each 3-carbon molecule oxidised to produce 2 ATP molecules and 1 NADH)
    • Net gain of 2 ATP and 2NADH molecules
  5. Key regulators in glycolysis
    • Phosphofructokinase
    • pyruvate kinase
    • pyruvate dehydrogenase
    • (regulate the rate of glycolysis process)
  6. Glycolysis (Stage I)
    • Hexokinase phosphorylates glucose (G) to G-6P (1 ATP used)
    • Phosphoglucose isomerase isomerises G-6P to fructose (F)-6P (6 to 5 member ring conversion)
    • Phosphofructokinase (PFK) phosphorylates F-6P to fructose 1,6-bisphosphate (F-1,6BP) (1 further ATP used)
  7. Glycolysis (Stage II)
    • F-1,6BP is split into isomers, DHAP and GAP (interchangable), by aldolase (highly reversible)
    • DHAP converted reversibly to GAP by triose phosphate isomerase, hence 1 glucose gives 2 GAP molecules
  8. Glycolysis (stage III)
    • GAP dehydrogenase (GAPDH) catalyses oxidation of GAP to 1,3-bisphoshoglycerate (1,3-BPG) with production of NADH and H+ (reduced)
    • 1,3-BPG has high phosphoryl transfer potential. Phosphoglycerate kinase transfers phosphate group to ADP to form 1 ATP molecule
    • 3-PG is converted to 2-PG by Phosphoglycerate mutase
    • 2-PG is dehydrated to phosphoenol-pyruvate (PEP) by enolase
    • high phosphoryl transfer potential of PEP catalysed to pyruvate by pyruvate kinase (PK) with formation of 1 ATP
  9. Glycolysis net yield
    • Glucose to pyruvate net yield: Glucose + 2Pi + 2 ADP + 2 NAD+
    • --> 2 pyruvate + 2 ATP + 2 NADH + 2H+ + 2H2O
    • redox balance and outcome of pyruvate
    • reduction of pyruvate and acetaldehyde by NADH to lactate (LDH) and ethanol (ADH) respectively
    • (krebs cycle)- regeneration of NAD+
    • pyruvate transferred to mitochondra, decarboxylated and oxidised to form acetyl CoA
    • Pyruvate + NAD+ + CoA--> acetyl CoA + CO2 and NADH (pyruvate dehydrogenase complex)
  10. Cori cycle
    • in skeletal muscle undergoing intense contraction and in RBC (no mitochondria), pyruvate and NADH accumulate. NAD+ is regenerated by reduction of pyruvate to lactate (LDH)
    • lactate diffuses into circulation to liver. where it is oxidised back to pyruvate and subsequently converted into glucose to be fed back to muscle (allowing glycolysis to cont. w/o O2)
  11. Fructose enters glycolysis pathway at intermediate stages...
    • Fructose is converted by fructokinase to fructose 1-phosphate (F-1P). F-1P split into DHAP and GAP by another aldolase, F-1P aldolase. REQUIRES ATP (for their breakdown)
    • alternatively fructose converted (with less affinity than glucose) by hexokinase to F-6P. requires ATP
  12. Galactose enters glycolysis pathway at intermediate stages...
    • galactose is converted to glucose-6P in 4 steps
    • (galactokinase, galactose 1- phosphate uridyl transferase, epimerase, phosphoglucomutase)
    • Galactose + ATP---> glucose 6-phosphate + ADP + H+
  13. Milk intolerance
    • insufficient lactase in the gut to cope with milk intake
    • lactase breaks down lactose to glucose and galactose
    • post- weaning lactase levels down to 5 to 10% of birth
    • microbial breakdown of lactose to lactic acid, methane and hydrogen. undigested lactose and lactic acid osmotically draw fluid into gut lumen, hence diarrhoea
  14. Regulatory checkpoints of glycolysis
    • irreversible reactions!! of (regulatory checkpoints):
    • hexokinase (G --> G-6P)
    • Phosphofructokinase (PFK) (F-6P --> F-1,6BP)
    • Pyruvate kinase (PK) (PEP --> pyruvate)
    • the corresponding genes are regulated at the level of transcription and translation
    • their enzymatic activities are regulated by allosteric effects or by covalent modifications
  15. Regulation of PFK activity
    • PFK is a tetramer, each subunit with a catalytic and an allosteric site
    • -allosteric ATP binding to PFK reduces affinity of PFK to F-6P
    • -rise in [H+] or fall in pH inhibits PFK (as in lactic acid build up)
    • -Citrate, an early intermediate of the citric acid cycle, enhances the inhibitory effect of ATP
    • -Low glucose level
    • -AMP reverses the allosteric inhibition of ATP
    • -High Glucose level
    • -Allosteric F-2,6BP (produced in the liver) binding stimulates PFK (NOT F-1,6 BP)
  16. Allosteric activator: F-2,6 BP
    • Glucose high: PFK2 (dual kinase and phosphatase) converts F-6P to F-2,6BP, which in turn stimulates PFK (note PFK2 does not equal PFK)
    • Glucose Low: --> glucagon increases --> protein kinase A --> phosphorylation of PFK2 --> activation of it's phosphatase domain --> reduced F-2,6BP --> reduced PFK activity (in liver)
    • (glucagon promotes glycogenolysis and gluconeogenesis)
  17. Hexokinase
    • Hexokinase (G --> G-6P) is inhibited by G-6P
    • inhibition of PFK (F-6P --> F-1,6BP) will eventually lead to build up of G-6P. hence inhibition of PFK will also inhibit hexokinase
    • Role of isozyme glucokinase (activitaed w/ glucose levels are very high)(Gk, lower affinity for glucose and not inhibited by G-6P):
    • when glucose high, Gk provides G-6P for glycogen synthesis (G-6P can also be oxidised by pentose phosphate pathway to generate NADPH)
  18. Pyruvate Kinase (PK)
    • PK (PEP --> pyruvate + ATP), a tetramer, encoded by different genes to give rise to L-type (liver isoform) and M=-type (muscle and brain isoform)
    • Activators of PK:
    • -F-1,6BP (product of PFK) activates both PK isoforms
    • -insulin signalling leads to dephosphorylation (activation) of PK
    • Inhibitors of PK:
    • -ATP allosteric inhibition of both PK isoforms
    • -alanine (made from pyruvate) allosteric inhibition (abundance of building blocks)
    • -glucogon signalling leads to phosphorylation (inactivation) of L isoform of PK
  19. Regulation of rate of glycolysis at level of Pyruvate kinase (liver isoform)
    • high glucose (insulin) phosphorylated pyruvate kinase (less active)
    • dephosphorylated pytuvate kinase (active)->Phosphoenolpyruvate + ADP + H+-->Pyruvate + ATP
  20. Pyruvate kinase deficiency
    • an inherited autosomal disorder
    • reduced survival of red blood cells (no mitochondria and nucleus) from deficiency in ATP
    • loss of biconcave shape and haemolytic anemia
  21. Pentose phosphate pathway (PPP)
    • primarily anabolic. utilizes 6 carbons of glucose --> 5 carbon sugars and reducing equivalents (NADPH)
    • however, does oxidise glucose and under some conditions can completely oxidize glucose to CO2 and H2O
  22. PPP relationship to glycolysis
    PPP pathway branches from glycolysis at level of G-6-P
  23. primary functions of PPP
    • to generate reducing equivalents, in the form of NADPH, for reductive biosynthesis
    • to provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acid
  24. NADPH for reductive biosynthesis
    • in nucleated cells w/active lipid biosynthesis (eg lactating mammary glands, adrenal cortex and liver)
    • NADPH is used in redox rxns required for biosynthesis of fatty acids, cholesterol, steroid hormones, and bile salts
    • IN LIVER: NADPH used for hydroxylation rxns involved in detoxification and excretion of drugs
    • IN RBCs: NADPH used in reduction of glutathione
  25. PPP secondary function
    metabolize dietary pentose sugars (5 carbon atoms) derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates
  26. PPP shunt
    • when pentoses not needed for biosynthetic rxns, pentose phosphate intermediates are cycled back into mainstream of glycolysis by conversion in f-6P and glyceraldehyde-3-phosphate
    • (shunted back so nothing is wasted)