Transcript Chapter 16 Glycolysis and gluconeogenesis
Chapter 16 Glycolysis and
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gluconeogenesis
Glycosis is an energy-conversion pathway in many organisms
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The glycolytic pathway is tightly controlled
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Glucose can be synthesized from noncarbohydrate precursors
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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 (Man 4 ) 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
Stage 1 of glycolysis
p. 438 bis- vs. di Trap Glc * *
*
Aldose 6 ring
*
* * * Ketose 5 ring
Hexokinase: requires Mg 2+ or Mn 2+ 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
*
*
p. 427 lyase
*
*
*
isomerase *
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 k cat /K M : 2
10 8 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
×
10 7 international units
H of C1 H of C2 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.
TPI suppresses an undesired side rx.
Stage 3 of glycolysis
A high phosphoryl transfer potential
Carboxylic acid compound Two processes must be coupled high-energy compound
preserve energy
Aldehyde acid polarization p. 442 His176 Cys149 Hemithioacetal p. 306 NAD + 1 p. 420 NADH1 release NAD + 2 Energy released by carbon oxidation
High energy compound
reversible * * Substrtate-level phosphorylation Intracellular shift * * Substrtate-level phosphorylation * CO 2
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 P i + 2 ADP + 2 NAD +
2 Pyr + 2 ATP + 2 NADH + 2 H + + 2 H 2 O
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 O 2 .
Pyruvate
ethanol
in yeast and several organisms
thiamine pyrophosphate zinc ion Centrum
Glc + 2 P i + 2 ADP + 2 H +
2 ethanol + 2 ATP + 2 CO 2 + 2 H 2 O p. 446 (Fig. 16.10)
Pyruvate
lactate
occur in higher organisms, the amount of oxygen is limiting Glc + 2 P i + 2 ADP
2 Lactate + 2 ATP + 2 H 2 O Magnesium lactate: a gel constituent; inhibit the production of histamine by histidine decarboxylase
lactose
Obligate anaerobes: – organisms cannot survive in the presence of O 2 Facultative anaerobes: organisms can function in the presence or absence of O 2 CAM
via microorganisms Watermelon juice: facilitate ethanol biofuel production Biotech. for Biofuels (2009) 2: 18
p. 449
NAD
+
binding region in dehydrogenase
G3P dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase
Rossmann fold
4 helices 6 parallel sheet N icotinamide a denine d inucleotide
Entry point in glycolysis of galactose fructose and
Fructose metabolism
(liver)
hexokinase
F 6-P (adipose tissue)
affinity compartment
2ATP
Galactose metabolism
hexokinase
Galactose metabolism
Polysaccharides Glycoproteins p. 314
G6P
mutase
Lactose intolerance (hypolactasia)
– a deficiency of
lactase
(2) - lactase 3 lactic acid + 3 CH 4 + H 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.
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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)
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.
) (Hyperbolic) (sigmoid) ¤ [ 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 Km
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 70-115 mg/100 ml endurance exercise, GLUT4 No.
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 Triacylglycerol hydrolysis
protein breakdown
active skeletal muscle
- 7.5 kcal/mol
G
°´
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
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The stoichiometry of Glycolysis vs. Gluconeogenesis Glycolysis:
Glucose + 2 ADP + 2 P i
+ 2 NAD 2 Pyr + 2 ATP + 2 NADH + 2H + +
G 0’ = - 20 kcal / mol + 2 H 2 O
if reverse?
¤
Gluconeogenesis:
2 Pyr + 4 ATP
+ 2 GTP +
G 0’ = - 9 kcal / mol 2 NADH + 6 H Glucose + 4 ADP + 2 GDP + 6 P i 2 O + 2 NAD + + 2H + NTP hydrolysis is used to power an energetically unfavorable reaction Both reactions are exergonic
Compartmental cooperation - mitochondrial
Pyruvate carboxylase Mito NADH-malate dehydrogenase
G 0’ decarboxylation PEP + CO 2 GT P
PEP carboxykinase
Specific transporter
NAD + -malate dehydrogenase
Pyruvate carboxylase
(Pyr + CO 2 + ATP + H 2 O
OAA + ADP + P i + 2 H + )
The only mitochondrial enzymes among the enzymes of gluconeogenesis
(ATP-activating domain, p. 711) HCO 3 Carbonic anhydrase + ATP
HOCO 2 -PO 3 2 + ADP carboxyphosphate: activated form of CO 2 Biotin-Enz + HOCO 2 PO 3 2-
CO 2 -biotin-Enz + Pi is activated by acetyl CoA (p. 493) CO 2 -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:
Glycolysis and gluconeogenesis are not time highly active at the same – 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
Biofunctional of phosphofructokinase 2 phosphofructokinase / fructose bisphosphatase 2 F6P
F2,6BisP a single 55-kd polypeptide chain
Janus
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 K m of sugars: 0.01 ~ 0.1 mM Glucokinase : not inhibited by G6P K m 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 Ala + NADH Ala transaminase + NAD + Ala metabolism: maintain nitrogen balance
carriers
Pyr Absence of O 2 TCA cycle Well-oxygenated Lactate
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 ¤ H 4 isozyme (type 1): high affinity for lactate, lactate
pyruvate, under aerobic condition H 3 M 1 H 2 M 2 isozyme (type 2) isozyme (type 3)
H 1 M 3 M 4 isozyme (type 4) 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