Chapter 16 Glycolysis and gluconeogenesis

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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

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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
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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
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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 5 ring

p. 438 bis- vs. di-


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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. 221-222
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
His stabilize the negative charge that develops
on the C-2 carbonyl group
H of C1
methyl glyoxal Pi
H of C2
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
Two processes must be coupled
high-energy compound ? preserve energy
Carboxylic acid compound
17
His176
NAD1
Aldehyde
Hemithioacetal p. 306
Cys149
p. 420
polarization
p. 442
NAD2
NADH1 release
acid
Energy released by carbon oxidation ? High energy
compound
18

reversible
Substrtate-level phosphorylation Intracellular
shift Substrtate-level phosphorylation

CO2


19
3 phosphoglycerate ? 2 phosphoglycerate Enz-His-ph
osphate 3 phosphoglycerate ? Enz-His
2,3-bisphosphoglycerate Enz-His
2,3-bisphosphoglycerate ? Enz-His-phosphate 2
phosphoglycerate
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• Glc 2 Pi 2 ADP 2 NAD
• 2 Pyr 2 ATP 2 NADH 2 H 2 H2O

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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.
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Pyruvate ? ethanolin 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. 16.10)
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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
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NAD binding region in dehydrogenaseG3P
dehydrogenase, alcohol dehydrogenase, lactate
dehydrogenase
p. 449
Rossmann fold 4 ? helices 6 parallel ? sheet
Nicotinamide adenine dinucleotide
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Entry point in glycolysis of galactose and
fructose
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Fructose metabolism
hexokinase
(liver)
F 6-P
(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.
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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

Glycolysis in muscle ? is controlled by energy
charge ? Phosphofructokinase is the most
important control site in glycolysis
F6P?F1,6bisP homotetramer
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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
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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
Glycolysis in liver liver function
maintains blood-glucose level, the regulation is
more complex than muscle Phosphofructokinas
e ? 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
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
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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
70-115 mg/100 ml
Normal serum-glucose level 48 mM
p. 457
endurance exercise, GLUT4 No. ?
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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
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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 ?
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?G
0.7 -0.5
- 7.5 kcal/mol
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Glycolysis vs. Gluconeogenesis

• Three irreversible reactions, irrespective
• Glycolysis
• hexokinase, phosphofructokinase, pyruvate
  kinase
• Gluconeogenesis
• glucose 6-phosphatase, fructose
  1,6-bisphosphatase,
• pyruvate carboxylase, phosphoenolpyruvate
  carboxykinase

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The stoichiometry of Glycolysis vs.
Gluconeogenesis

• Glycolysis
• Glucose 2 ADP 2 Pi 2 NAD
• ? 2 Pyr 2 ATP 2 NADH 2H 2 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

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Compartmental cooperation- mitochondrial
Pyruvate carboxylaseMito
NADH-malate dehydrogenase
?G0 decarboxylation
Specific transporter
NAD-malate dehydrogenase
GTP
PEP CO2
PEP carboxykinase
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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)
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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
Reciprocal controlGlycolysis and
gluconeogenesis are not highly active at the same
time
p. 465
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
2phosphofructokinase / fructose bisphosphatase
2F6P ? F2,6BisP
Janus
a single 55-kd polypeptide chain
L (liver) / M (muscle) isoforms
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Fructose 2,6-bisphosphate synthesis and
degradation
PEP carbokinase ? F 1,6-bisphosphatase
? Glycolytic enzymes ? (pyruvate kinase)
In liver
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The first irreversible reaction of
glycolysisGlc ? 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

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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
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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
• honeybeesonly PFKase (02)

malignant hyperthermia
If ? 10?
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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
Well-oxygenated
TCA cycle
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Integration of glycolysis and gluconeogenesis
during a sprint
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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.

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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
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96T2
96T3
97T
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97T
98T
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98T
70
98T
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96C
97C
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