1. Chapter 16 Glycolysis and gluconeogenesis
§ Glycosis is an energy-conversion pathway in many organisms
§ The glycolytic pathway is tightly controlled
§ Glucose can be synthesized from noncarbohydrate precursors
§ Gluconeogenesis and glycolysis are reciprocally regulated

2. Glucose fates Glucose: is an important fuel for most organisms
the only fuel that the brain uses under nonstarvation conditions
the only fuel that red blood cells can use at all
almost all organisms exist a similar process for glucose
p. 435 speculate the reasons

3. Lactate fermentation in muscle extracts
A key discovery was made by Hans Buchner and Eduard Buchner in 1897, quite by accident.
 To manufacture cell-free extracts of yeast for possible therapeutic use,
replace phenol
 Try sucrose (non-reducing sugar), sucrose was rapidly fermented into alcohol
by the yeast juice, sucrose fermentation
 Fermentation could take place outside living cells
1860 Louis Pasteur: fermentation is inextricably tied to living cells.
 Open the door to modern biochemistry
Lactate fermentation in muscle extracts
Glycosis is known as the Embden-Meyerhof pathway

4. Glucose is generated from dietary carbohydrates
is an important fuel for most organisms
Starch and glycogen:
are digested by -amylase released by pancreas and saliva. The products
are maltose and maltotriose and the undigested product, limit dextrin.
Maltase, -glucosidase, -dextrinase
Sucrase, lactase
Synthesis high mannose type oligosaccharide to develop HIV-1 vaccine (Man4)
Chen CY, Wong CH (2007) Master thesis, NTU
The side-effects of anti-reverse transcriptase

5. § 16.1 Glycolysis – an energy-conversion pathway
 three stages
1. consume energy
2. 6C is cleaved into 2 phosphorylated 3C
3. energy production
– takes place in the cytoplasm
invest

6. * * * * * * * Stage 1 of glycolysis Aldose 6 ring Trap Glc Ketose
p. 438 bis- vs. di- * *

7. Hexokinase: requires Mg2+ or Mn2+
Other kinase  to form a complex with ATP
12
On Glc binding
 Conformation markedly change
except the – OH of C6 is not
surrounded by protein,
phosphorylation

8. * * * *
isomerase * *
p. 427 lyase

9. * * * * Stage 2 of glycolysis F1,6-bisP TPI or TIM
major in equilibrium
The subsequent reaction remove G3P

10. TPI structure:  8 parallel  strands surrounded by 8  helices
 a general acid-base rx.
 Glu 165, His 95
 a kinetically perfect enzyme
kcat/KM: 2  108 M-1 s-1
close to the diffusion-controlled limit p

11. One international unit of enzyme:
the amount that catalyzes the formation of 1 mole of production in 1 min.
the conditions of assay must be specified.
Katal:
one katal is that amount of enzyme catalyzing the conversion of 1 mole of
substrate to product in 1 sec.
 1 katal = 6 × 107 international units

12. The active site is kept closed until the desired rx. takes place.
His stabilize the negative charge that develops on the C-2 carbonyl group
H of C1
H of C2
methyl glyoxal + Pi
The active site is kept closed until the desired rx. takes place.

13. TPI suppresses an undesired side rx.

14. Stage 3 of glycolysis

15. A high phosphoryl-transfer potential

16. Carboxylic acid compound
Two processes must be coupled
high-energy compound
 preserve energy
Carboxylic acid compound

17. p. 420 Cys149 acid Energy released by carbon oxidation
His176
NAD+1
Aldehyde
Hemithioacetal
p. 306
Cys149
p. 420
polarization
p. 442
NADH1 release
NAD+2
acid
Energy released by carbon oxidation
 High energy compound

18. * * *CO2 * * reversible Substrtate-level phosphorylation
Intracellular shift
*CO2 * *

19. 3 phosphoglycerate  2 phosphoglycerate
Enz-His-phosphate + 3 phosphoglycerate  Enz-His + 2,3-bisphosphoglycerate
Enz-His + 2,3-bisphosphoglycerate  Enz-His-phosphate + 2 phosphoglycerate

21. Glc + 2 Pi + 2 ADP + 2 NAD+
2 Pyr + 2 ATP + 2 NADH + 2 H+ + 2 H2O

22. The diverse of fates of pyruvate
Labeling isotope C3, C4
recycling
Fermentation:
An ATP-generating process in which organic compounds act as both donors and acceptors of electrons. Fermentation can take place in the absence of O2.

24. Pyruvate  ethanol in yeast and several organisms
thiamine pyrophosphate
zinc ion
Centrum
Glc + 2 Pi + 2 ADP + 2 H+  2 ethanol + 2 ATP + 2 CO2 + 2 H2O
p. 446 (Fig )

25. Pyruvate  lactate occur in higher organisms, the amount of oxygen is limiting
lactose
Glc + 2 Pi + 2 ADP  2 Lactate + 2 ATP + 2 H2O
Magnesium lactate: a gel constituent; inhibit the production of
histamine by histidine decarboxylase

26. Obligate anaerobes: – organisms cannot survive in the presence of O2
Facultative anaerobes:
organisms can function in the presence or absence of O2
CAM

27. via microorganisms
Watermelon juice: facilitate ethanol biofuel production
Biotech. for Biofuels (2009) 2: 18

28. NAD+ binding region in dehydrogenase G3P dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase
Rossmann fold
4  helices
6 parallel  sheet
Nicotinamide adenine dinucleotide

29. Entry point in glycolysis of galactose and fructose

30. Fructose metabolism hexokinase (liver) F 6-P 2ATP (adipose tissue)
affinity
compartment
2ATP

31. Galactose metabolism
hexokinase

32. Galactose metabolism
p. 314
Polysaccharides Glycoproteins
mutase
G6P

33. Lactose intolerance (hypolactasia) – a deficiency of lactase
(2)
- lactase
3 lactic acid + 3 CH4 + H2
Osmotic induction  diarrhea

34. Galactosemia: an inherit disease
– galactose 1-phosphate uridyl transferase deficiency, diagnostic criterion for red blood cells
– diarrhea, liver enlargement, jaundice and cirrhosis, cataracts, lethargy, retarded mental development
– a delayed acquisition of language skills, ovarian failure for female patients
p. 452 There is a high incidence of cataract formation with age in populations that consume substantial amounts of milk into adulthood.

35. § 16.2 The glycolytic pathways is tightly controlled
 essentially irreversible reactions, three reactions
 The methods of enzyme activity regulation
allosteric effector ~ ms
covalent phosphorylation ~ s
transcription ~ h
 A dual role of glycolysis:
generate ATP and provide building blocks, such as fatty acid synthesis
 Skeletal muscle and liver regulation (Ch. 21)

36.  is controlled by energy charge
Glycolysis in muscle:
 is controlled by energy charge
 Phosphofructokinase is the most important control site in glycolysis
F6PF1,6bisP
homotetramer –

37. Phosphofructokinase – allosteric regulation
 energy charge, ATP / AMP (,  PFKase act. )
 pH value ( pH focus at lactic acid  PFKase act.  )
¤ [AMP] is positive
regulator
¤ adenylate kinase
2 ADP  ATP + AMP
ATP is salvaged from ADP
¤ total adenylate pool is
constant
[ATP] [ADP] [AMP]
ex. 15
(Hyperbolic)
(sigmoid) Km

38. Hexokinase: is inhibited by its product, G6P
Glycolysis in muscle:
Hexokinase: is inhibited by its product, G6P
G6P fates (Ch. 20)
increase [G6P] imply:
no longer requires Glc for energy or for the synthesis of glycogen
 Glc will be left in the blood
if phosphofructokinase is inhibited  [F6P] 
 [G6P]   hexokinase is inhibited
Pyruvate kinase:
is allosterically inhibited by ATP and alanine, former is related to energy
charge and latter is building blocks

39. Glycolysis in muscle:

40. liver function: maintains blood-glucose level, the regulation is more
Glycolysis in liver:
liver function: maintains blood-glucose level, the regulation is more
complex than muscle
Phosphofructokinase:
 inhibited by citrate [TCA cycle] and enhancing the inhibitory effect of ATP
(not by pH of lactate)
 activated by fructose 2,6-bisphosphate (F 2,6-BP)
[Glc]  [F 2,6-BP]   glycolysis  [feedforward stimulation]

41. Phosphofructokinase – activated by fructose 2,6-bisphosphate

42. liver function: maintains blood-glucose level
Glycolysis in liver:
liver function: maintains blood-glucose level
Glucokinase replace hexokinase
Glucokinase
is not inhibited by glucose 6-phosphate
provide glucose 6-phosphate for the synthesis of glycogen and
for the formation of fatty acid
its affinity for glucose is about 50-fold lower than that of hexokinase
 brain and muscle first call on glucose when its supply is limited.
P. 456

43. Glycolysis in liver: Pyruvate kinase: – a tetramer of 57 kd subunits
– isozymic forms: Liver (L) are controlled by reversible phosphorylation
Muscle and brain (M)
Glucagon 
cAMP
Protein kinase A
Allosteric inhibition
Isozymes contribute to the metabolic diversity of different organs

44. Glucose transporters: enable glucose to enter or leave animal cells
mg/100 ml
p. 457
Normal serum-glucose level: 4~8 mM
endurance exercise, GLUT4 No. 

45. Hypoxia-inducible transcription factor (HIF-1)
– increase the expression of most glycolytic enzymes and glucose transporters
– increase the expression of vascular endothelial growth factor (VEGF)
angiogenic factors
Anaerobic exercise, activate HIF-1, ATP generation
Cancer stem cells
anoxia
Hypoxia vs. menstrual cycle HIF

47. Gluconeogenesis  is not a reversal of glycolysis
 noncarbohydrate precursors of Glc, carbon skeleton
 take place in liver, minor in kidney, brain, skeletal and heart muscle, to maintain the Glc level in the blood
 Glc is the primary fuel of brain, and the only fuel of red blood cells
Triacylglycerol
hydrolysis 
protein breakdown 
active skeletal muscle 

51. G°´
0.7
-0.5
- 7.5 kcal/mol

52. Glycolysis vs. Gluconeogenesis
¤ Three irreversible reactions, irrespective
Glycolysis:
hexokinase, phosphofructokinase, pyruvate kinase
Gluconeogenesis:
glucose 6-phosphatase, fructose 1,6-bisphosphatase,
pyruvate carboxylase, phosphoenolpyruvate carboxykinase

53. The stoichiometry of Glycolysis vs. Gluconeogenesis
Glucose + 2 ADP + 2 Pi + 2 NAD+
 2 Pyr + 2 ATP + 2 NADH + 2H H2O
G0’= - 20 kcal / mol
if reverse?
¤ Gluconeogenesis:
2 Pyr + 4 ATP + 2 GTP + 2 NADH + 6 H2O
 Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ + 2H+
G0’= - 9 kcal / mol
NTP hydrolysis is used to power an energetically unfavorable reaction
Both reactions are exergonic

54. Compartmental cooperation - mitochondrial
Pyruvate carboxylaseMito
NADH-malate dehydrogenase
G0’
decarboxylation
Specific transporter
NAD+-malate dehydrogenase
GTP
PEP + CO2
PEP carboxykinase

55. Pyruvate carboxylase (Pyr + CO2 + ATP + H2O OAA + ADP + Pi + 2 H+)
The only mitochondrial enzymes among the enzymes of gluconeogenesis
(ATP-activating
domain, p. 711)
Carbonic anhydrase
HCO3- + ATP  HOCO2-PO32- + ADP carboxyphosphate: activated form of CO2
Biotin-Enz + HOCO2-PO32-  CO2-biotin-Enz + Pi is activated by acetyl CoA (p. 493)
CO2-biotin-Enz + Pyr  biotin-Enz + OAA S
-amino group of Lys
(PCase)

56. Free glucose generation
F1,6bisP  F6P  G6P •••  Glc
The endpoint of gluconeogenesis in most tissues,
can keep Glc or G6P is converted into glycogen.
In liver and to a lesser extent the kidney,
five proteins are involved
(Does not take place in cytoplasm)
SP: a calcium-binding stabilizing protein
Gluconeogenesis 

57. p. 465
Reciprocal control: Glycolysis and gluconeogenesis are not highly active at the same time
– Energy state
– Intermedia:
allosteric effectors
– Regulators:
hormones
 Amounts and activities
of distinctive enzymes 
Starvation:
glucagon
rich in precursors
high energy state
Fed state:
insulin
low energy state 

58. Biofunctional of phosphofructokinase 2 phosphofructokinase / fructose bisphosphatase 2 F6P  F2,6BisP
Janus
a single 55-kd polypeptide chain
L (liver) / M (muscle) isoforms

59. Fructose 2,6-bisphosphate: synthesis and degradation
PEP carbokinase 
F 1,6-bisphosphatase 
Glycolytic enzymes 
(pyruvate kinase)
In liver:

60. The first irreversible reaction of glycolysis: Glc  G6P
¤ Hexokinase: is inhibited by G6P
Km of sugars: 0.01 ~ 0.1 mM
Glucokinase: not inhibited by G6P
Km of glucose: ~10 mM
present in liver, to monitor blood-glucose level.
¤ Committed step
the most important control step in the pathway
G6P glycogen biosynthesis
 fatty acid biosynthesis
 pentose phosphate pathway

61. Hormones ¤ Affect the expression of the gene of the essential enzymes
– change the rate of transcription
– regulate the degradation of mRNA
¤ allosteric control (~ms); phosphorylation control (~ s);
transcription control (~ h to d)
The promoter of the PEP carboxykinase (OAAPEP) gene
IRE: insulin response element;
GRE: glucocorticoid response element
TRE: thyroid response element
CRE: cAMP response element

62. Substrate cycle (futile cycle)
Biological significances
Simultaneously fully active
(1) Amplify metabolic signals
(2) Generate heat
bumblebees:
PFKase
F1,6-bisPTase:
is not inhibited by AMP
honeybees: only PFKase (02)
malignant hyperthermia
If  10

63. Cori cycle:
Contracting skeletal muscle supplies lactate to the liver, which uses it to synthesize and release glucose
+ NADH
Ala
Ala
transaminase
+ NAD+
carriers
Absence of O2
Pyr
Lactate
Ala metabolism:
maintain nitrogen balance
TCA cycle
Well-oxygenated

64. Integration of glycolysis and gluconeogenesis during a sprint

65. Lactate dehydrogenase
¤ a tetramer of two kinds of 35-kd subunits encoded by similar genes
¤ H type: in heart (muscle)
M type: in skeletal muscle and liver
¤ H4 isozyme (type 1): high affinity for lactate, lactatepyruvate,
under aerobic condition
H3M1 isozyme (type 2)
H2M2 isozyme (type 3)
H1M3 isozyme (type 4)
M4 isozyme (type 5): pyruvate  lactate
under anaerobic condition
 a series of homologous enzymes,
foster metabolic cooperation between organs.

66. Avidin (Mr 70,000): rich in raw egg whites/a defense function
Ex. 11
Biotin: abundant in some foods and is synthesized by intestinal bacteria
Avidin (Mr 70,000): rich in raw egg whites/a defense function
                      
                                             
The Biotin-Avidin System can improve sensitivity because of
the potential for amplification due to multiple site binding.
Purification

67. 96T2
96T3
97T

68. 97T
98T

69. 98T

70. 98T

71. 96C
97C