Transcript Chapter 16 Glycolysis and gluconeogenesis

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
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
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
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
§ 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
Stage 1 of
glycolysis
*
Trap Glc
*
Aldose
6 ring
*
*
Ketose
5 ring
*
p. 438 bis- vs. di-
**
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
*
*
*
*
isomerase
*
*
p. 427 lyase
Stage 2 of
glycolysis
F1,6-bisP
*
*
TPI or TIM
*
*
major in equilibrium
The subsequent reaction remove G3P
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
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
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.
TPI suppresses an undesired side rx.
Stage 3 of glycolysis
A high phosphoryltransfer potential
Two processes
must be coupled
high-energy compound
Carboxylic acid
compound
 preserve energy
His176
NAD+1
Aldehyde
Cys149
Hemithioacetal
p. 306
p. 420
polarization
p. 442
NADH1 NAD+2
release
acid
Energy released by carbon oxidation
 High energy compound
*
reversible
*
Substrtate-level
phosphorylation
Intracellular shift
Substrtate-level
phosphorylation
*CO2
*
*
3 phosphoglycerate  2 phosphoglycerate
Enz-His-phosphate + 3 phosphoglycerate  Enz-His + 2,3-bisphosphoglycerate
Enz-His + 2,3-bisphosphoglycerate  Enz-His-phosphate + 2 phosphoglycerate
Glc + 2 Pi + 2 ADP + 2 NAD+
 2 Pyr + 2 ATP + 2 NADH + 2 H+ + 2 H2O
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.
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. 16.10)
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
Obligate anaerobes:
– organisms cannot survive in the presence of O2
Facultative anaerobes:
organisms can function in the presence or absence of O2
CAM
via microorganisms
Watermelon juice: facilitate ethanol biofuel production
Biotech. for Biofuels (2009) 2: 18
NAD+ binding region in dehydrogenase
p. 449
G3P dehydrogenase, alcohol dehydrogenase, lactate
dehydrogenase
Rossmann fold
4  helices
6 parallel  sheet
Nicotinamide adenine dinucleotide
Entry point in glycolysis of galactose and
fructose
Fructose metabolism
hexokinase
(liver)
F 6-P
(adipose tissue)
affinity
compartment
2ATP
Galactose metabolism
hexokinase
Galactose metabolism
p. 314
Polysaccharides
Glycoproteins
mutase
G6P
Lactose intolerance (hypolactasia)
– a deficiency of lactase
(2)
- lactase
3 lactic acid + 3 CH4 + H2
Osmotic induction  diarrhea
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.
§ 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)
Glycolysis in muscle:
 is controlled by energy charge
 Phosphofructokinase is the most important control site in glycolysis
F6PF1,6bisP
homotetramer
–
Phosphofructokinase – allosteric regulation
 energy charge, ATP / AMP (,  PFKase act. )
 pH value ( pH focus at lactic acid  PFKase act.  )
¤ [AMP] is positive
(Hyperbolic)
regulator
¤ adenylate kinase
2 ADP  ATP + AMP
ATP is salvaged from ADP
(sigmoid)
¤ total adenylate pool is
constant
[ATP] [ADP] [AMP]
Km
ex. 15
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
Glycolysis in muscle:
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]
Phosphofructokinase
– activated by fructose 2,6-bisphosphate
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
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
Glucose transporters:
enable glucose to enter or leave animal cells
p. 457
Normal serum-glucose level: 4~8 mM
endurance exercise, GLUT4 No. 
70-115 mg/100 ml
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
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
protein breakdown 
active skeletal muscle 
Triacylglycerol
hydrolysis

G°´
- 7.5 kcal/mol
0.7
-0.5
Glycolysis vs.
Gluconeogenesis
¤ Three irreversible reactions, irrespective
Glycolysis:
hexokinase, phosphofructokinase, pyruvate kinase
Gluconeogenesis:
glucose 6-phosphatase, fructose 1,6-bisphosphatase,
pyruvate carboxylase, phosphoenolpyruvate
carboxykinase
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
Compartmental cooperation
- mitochondrial
Pyruvate carboxylaseMito
NADH-malate dehydrogenase
G0’
decarboxylation
Specific transporter
NAD+-malate dehydrogenase
GTP
PEP + CO2
PEP carboxykinase
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
carboxyphosphate: activated form of CO2
HCO3- + ATP  HOCO2-PO32- + ADP
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)
Free glucose generation
F1,6bisP  F6P  G6P •••  Glc (Does not take place in cytoplasm)
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
SP: a calcium-binding stabilizing protein
Gluconeogenesis 
Reciprocal control:
p. 465
Glycolysis and gluconeogenesis are not highly active at the same
time
– Energy state
– Intermedia:
allosteric effectors
– Regulators:
hormones
 Amounts and activities
of distinctive enzymes
Fed state:

insulin
Starvation:
low energy state
glucagon

rich in precursors
high energy state
Biofunctional of phosphofructokinase 2
phosphofructokinase / fructose bisphosphatase 2
F6P  F2,6BisP
Janus
a single 55-kd polypeptide chain
L (liver) / M (muscle) isoforms
Fructose 2,6-bisphosphate: synthesis and degradation
PEP carbokinase 
F 1,6-bisphosphatase 
Glycolytic enzymes 
(pyruvate kinase)
In liver:
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
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
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
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
Ala metabolism:
maintain nitrogen balance
Pyr
TCA cycle
Lactate
Well-oxygenated
Integration of glycolysis and gluconeogenesis during a sprint
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.
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
96T2
96T3
97T
97T
98T
98T
98T
96C
97C