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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
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+
On Glc binding
Conformation markedly change
except the – OH of C6 is not
surrounded by protein,
phosphorylation
Other kinase to form a complex with ATP
12
*
*
*
*
p. 427 lyase
*
*
isomerase
Stage 2 of glycolysis
F1,6-bisP
***
*
major in equilibrium The subsequent reaction remove G3P
TPI or TIM
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
methyl glyoxal + Pi
The active site is kept closed until the desired rx. takes place.
H of C1
H of C2
TPI suppresses an undesired side rx.
Stage 3 of glycolysis
A high phosphoryl-transfer potential
high-energy compound
preserve energy
Two processes must be coupled
Carboxylic acid compound
Cys149
His176
Hemithioacetal
p. 306
NAD+1
NAD+2NADH1 release
polarization
Aldehyde
acid
Energy released by carbon oxidation
High energy compound
p. 442
p. 420
Substrtate-level phosphorylation
Intracellular shift
Substrtate-level phosphorylation
*
**CO2*
*reversible
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 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)
Pyruvate lactate occur in higher organisms, the amount of oxygen is limiting
Glc + 2 Pi + 2 ADP 2 Lactate + 2 ATP + 2 H2O
Magnesium lactate: a gel constituent; inhibit the production of
histamine by histidine decarboxylase
lactose
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 dehydrogenaseG3P dehydrogenase, alcohol dehydrogenase, lactate
dehydrogenase
Nicotinamide adenine dinucleotide
Rossmann fold
4 helices
6 parallel sheet
p. 449
Entry point in glycolysis of galactose and fructose
Fructose metabolism
(liver)
2ATP
F 6-P
hexokinase
(adipose tissue)
affinity
compartment
Galactose metabolism
hexokinase
Galactose metabolism
Polysaccharides Glycoproteins
G6P
mutase
p. 314
Lactose intolerance (hypolactasia) – a deficiency of lactase
- lactase
3 lactic acid + 3 CH4 + H2
(2)
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)
–
F6PF1,6bisP
homotetramer
Glycolysis in muscle:
is controlled by energy charge
Phosphofructokinase is the most important control site in glycolysis
Phosphofructokinase – allosteric regulation
energy charge, ATP / AMP (, PFKase act. )
pH value ( pH focus at lactic acid PFKase act. )
(sigmoid)
(Hyperbolic)¤ [AMP] is positive
regulator
¤ adenylate kinase
2 ADP ATP + AMP
ATP is salvaged from ADP
¤ total adenylate pool is
constant
[ATP] [ADP] [AMP]
ex. 15Km
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
– 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
Glycolysis in liver:
Pyruvate kinase:
Isozymes contribute to the metabolic diversity of different organs
Normal serum-glucose level: 4~8 mMp. 457
Glucose transporters:
enable glucose to enter or leave animal cells
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
active skeletal muscle
protein breakdown
Triacylglycerol
hydrolysis
- 7.5 kcal/mol
0.7
-0.5
G°´
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
NADH-malate dehydrogenase
NAD+-malate dehydrogenase
Specific transporter
PEP + CO2
PEP carboxykinase
GTP
Pyruvate carboxylaseMito
G0’
decarboxylation
Pyruvate carboxylase (Pyr + CO2 + ATP + H2O OAA + ADP + Pi + 2 H+)
The only mitochondrial enzymes among the enzymes of gluconeogenesis
S
(PCase)
HCO3- + ATP HOCO2-PO3
2- + ADP carboxyphosphate: activated form of CO2
Biotin-Enz + HOCO2-PO3
2- CO2-biotin-Enz + Pi is activated by acetyl CoA (p. 493)
CO2-biotin-Enz + Pyr biotin-Enz + OAA
(ATP-activating domain, p. 711)
-amino group of Lys
Carbonic anhydrase
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
SP: a calcium-binding stabilizing protein
(Does not take place in cytoplasm)
Gluconeogenesis
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
Fed state:
insulin
low energy state
Starvation:
glucagon
rich in precursors
high energy state
p. 465
Biofunctional of phosphofructokinase 2phosphofructokinase / fructose bisphosphatase 2
F6P F2,6BisP
L (liver) / M (muscle) isoforms
a single 55-kd polypeptide chain
Janus
Fructose 2,6-bisphosphate: synthesis and degradation
In liver:
PEP carbokinase
F 1,6-bisphosphatase Glycolytic enzymes (pyruvate kinase)
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 (OAAPEP) gene
IRE: insulin response element;
GRE: glucocorticoid response element
TRE: thyroid response element
CRE: cAMP response element
Substrate cycle (futile cycle)
Biological significancesSimultaneously fully active(1) Amplify metabolic
signals(2) Generate heat bumblebees: PFKase F1,6-bisPTase: is not inhibited by AMP
honeybees:only PFKase (02)
If 10
malignant hyperthermia
Cori cycle:
carriers
+ NADH
+ NAD+
AlaAla
Ala metabolism:
maintain nitrogen balance
transaminase
Contracting skeletal muscle supplies lactate to the liver, which uses it to synthesize and release glucose
Pyr LactateAbsence of O2
Well-oxygenatedTCA cycle
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, lactatepyruvate,
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.
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
Ex. 11
96T2
96T3
97T
97T
98T
98T
98T
96C
97C
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