Glucose Catabolism Ch. 14

1
Glucose Catabolism Ch. 14

• Overview
• Reactions
• 1-5 Energy invested, 6-10 energy recouped
• Fermentation
• Anaerobic end products Ethanol or Lactate
• Control - Substrate cycle PFK vs FBPase
• Other Hexoses - Fructose, galactose, mannose
• Pentose Phosphate pathway
• Produces NADPH and nucleotide sugars

2
Overview

• Glucose 2NAD 2ADP 2Pi -gt 2 Pyruvate
  2NADH 2ATP 2 H2O 4H
• The C6 sugar is phosphoryated twice (- 2 ATP)
• Cleaved to give two 3 carbon units
• Oxidized 2(NAD -gt NADH)
• 2 phosphoryl groups from each of 2 C3 units
  transferred to ADP (4 ATP)

3
Glycolysis Reactions 1-5

• 1. Hexokinase
• Glucose ATP -gt Glucose-6-phosphate ADP Pi
• 2. Phospho Glucose Isomerase
• G6P -gt Fructose-6-phosphate
• 3. Phospho Fructokinase
• F6P ATP -gt Fructose 1,6-bisphosphate ADP Pi
• 4. Aldolase
• FBP -gt Glyceraldehyde-3-phosphate
  Dihydroxyacetone phosphate
• 5. Triose Phosphate Isomerase
• DHAP ltgt GAP

4
Hexokinase

• Glucose ATP -gt Glucose-6-phosphate ADP Pi
• ATP supplied as Mg2 ATP
• Active site cleft closes over substrates water
  excluded

5
Phospho Glucose Isomerase (PGI)
CH2OPO32-
CH2OH
O
O
H
H
OH
H
OH
OH
OH
H
H

• Glucose-6-phosphate -gt Fructose-6-phosphate
• General Acid protonates O - ring opens
• Enediolate (C1 - C2) intermediate
• Enol -gt Keto at C2
• Ring closure by attack of O on C2 CO

6
Phospho Fructokinase (PFK)
CH2OPO32-
CH2OPO32-
O
OH
H
H

• F6P ATP -gt Fructose 1,6-bisphosphate ADP Pi
• Second input of ATP
• Mechanism similar to hexokinase
• Rate determining step in glycolysis
• Allosterically regulated by AMP, ATP and citrate

7
Aldolase
CH2OPO32- C O HO C H H C OH H C OH
CH2OPO32-
CH2OPO32- C O HO C H H C OH H C OH
CH2OPO32-
DHAP
GAP

• FBP -gt Dihydroxyacetone phosphate
  Glyceraldehyde-3-phosphate
• DHAP GAP numbered from center
• C3 bears the phosphoryl group
• Schiff base or metal ion catalysed reaction

8
Triose Phosphate Isomerase (TIM)

• DHAP ltgt GAP Keq 0.0473
• Only GAP utilized in subsequent reactions
• TIM is an a/b barrel, highly efficient
• Enediolate intermediate mechanism

9
Glycolysis Reactions 6-10

• 6. Glyceraldehyde-3-Phosphate Dehydrogenase
• GAP NAD Pi -gt 1,3-Bisphosphoglycerate
  NADH H
• 7. Phosphoglycerate kinase
• 1,3-BPG ADP -gt 3-Phosphoglycerate ATP
• 8. Phosphoglycerate Mutase
• 3PG -gt 2-phosphoglycerate
• 9. Enolase
• 2PG -gt Phosphenolpyruvate H2O
• 10. Pyruvate Kinase
• PEP ADP -gt Pyruvate ATP

10
Glyceraldehyde-3-Phosphate Dehydrogenase(GAPDH)

• GAP NAD Pi -gt 1,3-Bisphosphoglycerate NADH
  H
• Inactivated by iodoacetate - Cysteine in active
  site
• 3H at C1 is transferred to NADH - direct hydride
  transfer
• Thio ester acyl enzyme intermediate is displaced
  by Pi
• Acyl phosphate is a high energy bond
• Overall the reaction is endergonic but removal of
  1,3BPG by PGKdrives the reaction forward

11
Phosphoglycerate kinase (PGK)
O C O- H C OH CH2OPO32-

• 1,3-BPG ADP -gt 3-Phosphoglycerate ATP
• First ATP synthesis step
• ADP and ATP in complex with Mg
• ?Gis -12 kJ M-1

12
Phosphoglycerate Mutase (PGM)
O C O- H C OPO32- CH2OH
O C O- H C OH CH2OPO32-

• 3PG -gt 2-phosphoglycerate
• PGM contains phospho-histidine
• Determined by 32P transfer from 3PG
• Enzyme first transfers PO3 to make 2,3BPG
• 2,3BPG intermediate occasionally dissociates
• 2,3BPG is an allosteric effector of hemoglobin
• Side reactions in erythrocytes convert 1,3 2,3
  BPG
• 2PG ?G hydrolysis -17.6 kJ /mol

13
Enolase
O C O- H C OPO32- CH2OH
O C O- C OPO32- CH2

• 2PG -gt Phosphenolpyruvate H2O
• Mg binds first
• Two-step dehydrogenation
• 1. Abstraction of proton from C2
• 2. Elimination of C3 -OH

14
Pyruvate Kinase
O C O- C O CH3
O C O- C OPO32- CH2

• PEP ADP -gt Pyruvate ATP
• 2nd ATP generating step
• Beta phosphoryl of ADP attacks P
• Enol pyruvate -gt keto form

15
Fermentation

• Anaerobic processes to recycle NAD
• For glycolysis to continue NADH (from GAPDH
  reaction) -gt NAD
• In muscle, lactate dehydrogenase converts
  Pyruvate NADH -gt Lactate NAD
• In yeast, pyruvate decarboxylase and alcohol
  deydrogenase convert pyruvate NADH to CO2
  Ethanol NAD

16
Lactate Dehydrogenase (LDH)
O C O- C O CH3
O C O- HO C H CH3

• Pyruvate NADH -gt Lactate NAD
• Hydride transfer to C2 of pyruvate
• Lactate (lactic acid) build-up lowers pH
• Low pH causes muscle fatigue

17
Pyruvate Decarboxylase
O C O- C O CH3
H C O CH3

• Pyruvate -gt Acetaldehyde CO2
• Thiamine pyrophosphate (TPP) cofactor
• Attack on Carbonyl carbon release of CO2
• Mechanism Fig 14-20

18
Alcohol Dehydrogenase (ADH)
H C O CH3

• Acetaldehyde NADH -gt Ethanol NAD
• Similar to LDH

19
Control

• Flux forward rate - reverse rate Jvf - vr
• At equilibrium Flux 0 vf vr
• For rate determining steps vf gtgt vr J vf
• J can be varied by
• Substrate concentration
• Allosteric control
• Covalent modification - eg. Phosphorylation
• Substrate cycles
• independent forward and reverse reactions
• Change in enzyme levels - Genetic control

20
Control of Glycolysis in Muscle

• Hexokinase, PFK and Pyruvate Kinase
• the only reactions in glycolysis with large -?G
• PFK is the major regulatory point
• ATP is a substrate and allosteric inhibitor
• Binds to inhibitor site when PFK is in the T
  state
• AMP binds to R state

21
Substrate cycling

• Fructose 1-6 bisphosphatase
• 1,6-Fructose Bisphosphate -gtFructose6P Pi
• Provides a pathway for reverse reaction of PFK
• Opportunity for greater regulatory range
• Net effect of the two reactions is ATP hydrolysis
  - exothermic reaction generates heat
• Thermogenesis important for insect flight and
  Obesity

22
Other Hexoses

• Fructose -gt F1P
• Fructokinase makes Fructose-1-P
• Galactose -gt UDP-Glu -gt Glu-1P -gt Glu-6P
• Galactokinase makes Galactose-1-P
• Gal-1P Uridylyl tranferase makes UDP-Gal
• UDP Gal-4- epimerase makes UDP-Glu
• Mannose
• Hexokinase, makes Man-6-P
• phophomannose isomerase Man-6-P -gtF6P

23
Pentose Phosphate pathway

• NADPH - Reductant distinct from NADH
• NAD/NADH 1000
• NADP/NADPH 0.01
• 3G6P 6NADP 3H2O gt 6NADPH 6H 3CO2
  2F6P GAP
• 3 stages
• Oxidations to give NADPH and Ru5P
• Isomerizations to give R5P and Xu5P
• Bond breaking and making to give F6P and GAP

24
NADPH production

• G6P dehydrogenase
• G6P NADP gt 6-phosphoglucono-d-lactone NADPH
  H
• Phosphogluconolactonase
• 6-phosphoglucono-d-lactonegt 6-phosphogluconate
• Phosphogluconate dehydrogenase
• 6-phosphogluconate NADP gt Ru5P NADPH CO2

25
Ribulose-5-phosphate

• Ribulose-5-phosphate isomerase
• Ru5P gt Ribose 5P
• R5P is a precursor for nucleotide synthesis
• Ribulose-5-phosphate epimerase
• Ru5P gt Xylulose 5P

26
CC bond cleavage

• Transaldolase
• R5P Xu5P gt S7P GAP 55 gt 73
• E4P Xu5P gt F6P GAP 45 gt 63
• Transketolase
• S7P GAP gt E4P F6P 73 gt 64

27
Control

• G6PDH is regulated by substrate
• NADP
• G6P dehydrogenase deficiency
• Most alleles have low protein stability
• Low NADPH leads to oxidation damage to
  erythrocytes
• Linked to malaria resistance