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Chapter 16
Glycolysis and Gluconeogenesis
Outline
16.1. Glycolysis Is an Energy-Conversion Pathway in Many
Organisms
16.2. The Glycolytic Pathway Is Tightly Controlled
16.3. Glucose Can Be Synthesized from Noncarbohydrate
Precursors
16.4. Gluconeogenesis and Glycolysis Are Reciprocally
Regulated
2
Trioses (three carbons)
•Monosaccharides, the simplest carbohydrates, are aldehydes (醛類) or ketones (酮類)
that have two or more hydroxyl groups (氫氧基;羥基)
•The smallest monosaccharides, composed of three carbon atoms, are
dihydroxyacetone and D- and L- glyceraldehyde
Contains a keto group
Contains a aldehyde group
•Simple monosaccharides with four, five, six, and seven carbon atoms are called
tetroses, pentoses, hexoses, and heptoses (suffix –ose means sugar)
Sugar Stereochemistry
Horizontal = forward
Most sugars
are in the D
form
Vertical = back
Fisher
projections:
OH group is in the right
Berg et al. Biochemistry
D isomer
L isomer
Sugar Stereochemistry
Asymmetric C
farthest from
the aldehyde
(or ketone)
group
D-glucose
Hemiacetal formation
•Open-chain forms of these sugars cyclize into rings. In general,
an aldehyde can react with an alcohol to form a hemiacetal
Hemiketal formation
•A ketone can react with an alcohol to form a hemiketal
Haworth Structure of Glucose
•For an aldohexose such as glucose, a single molecule provides both the
aldehyde and the alcohol : the C-1 aldehyde in the open-chain form of glucose
reacts with the C-5 hydroxyl group to form an intramolecular hemiacetal
•The resulting cyclic hemiacetal, a six-membered ring, is called pyranose
because of its similarity to pyran
Haworth Structure of Fructose
•The C-2 keto group in the open-chain form of a ketohexose, such as fructose,
can form an intramolecular hemiketal by reacting with either the C-6 hydroxyl
group to form a six-membered cyclic hemiketal or the C-5 hydroxyl group to
form
a five-membered cyclic hemiketal
•The five-membered ring is called a furanose because of its similarity to furan
Some fates of glucose
•Glycolysis 糖解作用: metabolizes one molecule of glucose to two
molecules of pyruvate with the concomitant net production of
two molecules of ATP.
– The process is anaerobic, O2 is not required
– Pyruvate is further processed:
•Anarobically through fermentation 發酵作用, two molecules of ATP.
•Aerobically by complete oxidation to CO2, generating more ATP
Alcoholic fermentation : glucose to ethanol
2ATP
6C
2ATP
More ATP
2 x 3C
Lactic acid fermentation : glucose to lactate
10
Glycolysis
• Glycolysis:
• Convert one glucose to two
pyruvate, producing two ATP and
two NADH
• Derived from the Greek stem glyk, “sweet”; and the word lysis,
“dissolution”
• also known as the EmbdenMeyerhof pathway
• Common to virtually all cells
• In eukaryotes, occurs in
cytoplasm
• 2 stages, 10 steps
11
Gluconeogenesis
糖質新生
• Glucose can be synthesized from
noncarbohydrate precursors,
such as pyruvate and lactate, in
the process of gluconeogenesis
• Glycolysis and gluconeogenesis
have some of the same enzymes
in common, the two pathways
are not simply the reverse of each
other. The highly exergonic,
irreversible steps of glycolysis are
bypassed in gluconeogenesis.
12
Glucose is generated from dieatry
carbohydrates
Starch
Complex carbohydrate
Glycogen (肝醣)
Pancreatic α-amylase (澱粉酶)
Salivary α-amylase
(Amylase cleaves the α -1,4 bonds,
but not α -1,6 bond)
Maltose
Maltotriose simpler carbohydrate
Maltase
Maltose  two glucoses
(麥芽三糖)
α-glucosidae Digest maltotriose and others
Limit dextrin
(極限糊精; 指上述無法再
被分解之α-1,6 bond 的糖)
α -Dextrinase Digest limit dextrin
Sucrase
Sucrose  fructose + glucose
lactase
Lactose  glucose + galactose
monosaccharides
Absorption by the intestine
Transport in the blood
13
16.1. Glycolysis Is an Energy-Conversion
Pathway in Many Organisms
•Glycolysis is common to all cells, both prokaryotic and
eukaryotic.
– Take place in the cytoplasm
– Divided into two stage
•The trapping and preparation phase (Energy investment phase)
– Conversion of glucose into fructose 1,6-biphosphate
– Consists of three step
» Phosphorylation
» Isomerization
» Second phosphorylation
– The cleavage of fructose 1,6-bisphosphate into two
glyceraldehyde 3-phosphate
•ATP is generated (energy payoff phase)
– glyceraldehyde 3-phosphate are oxided to pyruvate
14
First stage of glycolysis
15
Mg2+
Mg2+
2 molecules
Fig 16.2 Stages of glycolysis
16
glyceraldehyde 3-phosphate
Mg2+
Mg2+
Mg2+
K+
Mg2+
K+
Fig 16.2 Stages of glycolysis
17
Hexokinase traps glucose in the cell and begins
glycolysis
• Glucose enters cells through specific transport proteins
• It is phosphorylated by ATP to form glucose 6phosphate
– G-6P cannot pass through the membrane
– Phosphoryl group destabilized glucose, thus facilitating
metabolism
Mg2+
一般而言, kinase的
反應大都需要Mg2+
此反應大致屬不可逆反應
18
8Å
• Glucose induced hexokinase
structural changes
• Substrate-induced cleft closing is
general feature of kinase
Glucose 與hexokinase結合後會使酵素更靠近(induced fit),
glucose周圍環境更極性，促使glucose與ATP反應，同時也
避免與水分子接觸，而阻擾ATP的反應。
Fig 16.3 Induced fit in hexokinase
19
Fructose 1,6-bisphosphate is generated from
glucose 6-phosphate
• The isomerization of glucose 6-phophate to fructose
6-phosphate
– Conversion of an aldose into ketose
– Catalyzed by phosphoglucose isomerase
– Reversible reaction
1
6
2
1
1
Enzyme binding
(His) and open
the ring
Aldehyde group
Dissociation and
closing of the ring
2
Keto group
20
• Fructose 6-phosphate is phosphorylated at the
expense of ATP to fructose 1,6-bisphosphate
– Catalyzed by phosphofructokinase (PFK)
• Play a central role in the metabolism
• 當ATP缺乏或ADP、AMP增多時，此酵素活性會被活化
Mg2+
只要形成F-1,6-BP, 意味反應只進入glycolysis
•bis- means two separate monophosphoryl groups
•di- means two phosphoryl groups are present and are connected by
an anhydride bond
21
The six-carbon is cleaved into two three-carbon
fragments
• Fructose 1,6 bisphosphate is cleaved into glyceraldehyde
3-phosphate (GAP) and dihydroxyacetone phosphate
(DHAP)
– Catalyzed by aldolase (醛縮酶)
1
2
3
4
5
6
Ketos
aldose
• At equilibrium, 96% DHAP
•GAP for the subsequent
reactions of glycolysis 22
Fig 12-5 The class I aldolase reaction.
23
Principles of Biochemistry Lehninger
Mechanism: trisoe phosphate isomerase slavages
a three-carbon fragment
Fig 16.4 structure of triose
phosphate isomerase
Fig 16.5 Catalytic mechanism of triose phosphate
isomerase
作用機制與glycolysis中 phosphoglucose isomerase相似
**Triose phosphate isomerase (TPI) has two noteworthy feature
• Kinetically perfect enzyme: 作用很快
24
• Suppresses an undesired side reaction: the active site is kept closed until
the desirable reaction takes place
The oxidation of an aldehyde to an acids powers
the formation of a compound with high
phosphoryl-transfer potential
1 molecule of glucose (6C)  2 molecule of GAP (3C)
• The conversion of glyceraldehyde 3phosphate into 1,3-bisphosphoglycerate
(1,3-BPG)
– Catalyzed by glyceraldehyde 3phosphate dehydrogenase
Energy payoff stage
High phosphoryl-transfer potential
25
Second stage of glycolysis
• Glyceraldehyde 3 –phosphate dehydrogenase
– Oxidation of the aldehyde to a carboxylic acid by NAD+
– Joining of the carboxylic acid and orthophosphate to
form the acyl-phosphate product.
26
Linked to the enzyme by
a thioester bond
不利反應進行
Fig 16.6 Free-energy profiles for glyceraldehyde oxidation followed by acylphosphate formation.
27
His 176
Cys 149
Positive charge polarized
the thioester intermediate
to facilitate the attack by
orthophosphate
Fig 16.8 Catalytic mechanism of glyceraldehyde28
3phosphate dehydrogenase
ATP is formed by phosphoryl transfer from 1,3bisphosphaoglycerate
Mg2+
Substrate-level phosphorylation is a type of chemical reaction that
results in the formation and creation of adenosine triphosphate (ATP)
by the direct transfer and donation of a phasphate group to
adenosine triphosphate (ADP) from a reactive intermediate.
29
rearrangement
dehydration
Enol phosphate has a high
phosphoryl-transfer potential
30
• Mutase: catalyzed the intramolecular shift of a chemical
group, such as a phosphoryl group
Enz-His-phosphate + 3-phosphoglycerate
 Enz-His + 2,3-bisphosphoglycerate
• Mutase functions as a phosphatase
Enz-His + 2,3-bisphosphoglycerate
 Enz-His-phosphate + 2-phosphoglycerate
Mg2+
31
Pyruvate kinase
Tautomerizes rapidly
and nonenzymatically
K+, Mg2+ or Mn2+
More stable ketone
32
Two ATP molecules are formed in the conversion
of glucose into pyruvate
The net reaction of gylcolysis:
Glucose + 2Pi + 2 ADP +2NAD+ 
2 pyruvate + 2ATP + 2 NDAH +2H+ + 2H2O
Energy release -96kJ/mol
33
NAD+ is regenerated from the
metabolism of pyruvate
Fermentation (absence of O2)
34
1. Alcohol fermentation
Ethanol is formed from pyruvate in yeast and several
other microorganisms
Require coenzyme
thiamine pyrophosphate (TPP)
and Mg2+
Glucose + 2Pi + 2 ADP +2H+ 
2 ethanol + 2CO2 + 2ATP + 2H2O
(No NAD+ and NADH in this equation)
35
2. Lactic acid fermentation
Lactate is formed from pyruvate in microoragnisms ,
higher organism (O2 limiting), as in muscle cells
(intense activity)
Glucose + 2Pi + 2 ADP 
2 lactate + 2ATP + 2H2O
36
37
The binding site for NAD+ is similar in many
dehydrogenase
Glyceraldehyde 3-phosphate
dehydrogenase
Alcohol dehydrogenase
Lactate dehydrogenase
•Different 3-D structure
•NAD+-binding site similar
Rossmann fold
Fig 16.12 NAD+-binding region in
dehydrogenases.
38
Fructose and galactose are converted
into glycolytic intermediates
•There are no catabolic
pathway for metabolizing
fructose or galactose
convert these sugars into a
metabolite of glucose
Fig 16.13 Entry points in glycolysis
for galactose and fructose
39
1. Fructose 1-phosphate pathway in liver
2. Fructose can be phosphorylated
to fructose 6-phosphate by
hexokinase  glycolysis
Fig 16.14 Fructose metabolism
40
Galactose is converted into glucose 6-phosphate in 4
steps:
• Galactose-glucose interconversion pathway
41
phosphoglucomutase
Glucose 6-phosphate
Glycolysis
Galactose + ATP 
42
glucose 1-phosphate + ADP + H+
Many adults are intolerant of milk because they
are different in lactase
• Lactose intolerance or hypolactasia 乳糖不耐症
– A deficiency of the enzyme lactase
• Lactose is a good energy for microorganisms in colon
and produce CH4 and H2
• Uncomfortable feeling of gut distension and flatulence
• diarrhea
在缺乏乳糖酶的情況下，人攝入的乳糖不能被消化吸收進血液，而是滯留在腸道。
腸道細菌發酵分解乳糖的過程中會產生大量氣體而造成腹脹、放屁。過量的乳糖還
會升高腸道內部的滲透壓，阻止對水分的吸收而導致腹瀉。 當未分解吸收的乳糖進
入結腸後，被腸道存在的細菌發酵成為小分子的有機酸如醋酸、丙酸、丁酸等，並
產生一些氣體如甲烷、H2、CO2等,這些產物大部分可被結腸重吸收。新生兒小腸粘
43
膜乳糖酶缺乏是主要病因，部分人群因長期不攝入奶及奶製品也會造成。
Galactosemia 半乳糖血症
• Inherited deficiency in galactose 1-phosphate uridyl
transferase activity
– 血中半乳糖濃度增加，並可於尿液中測出
– 出生時並無異狀，餵乳數天後發生嚴重吐奶、呈昏睡
狀，之後會有肝脾腫大、黃疸，嚴重的患者會因血液
感染而死亡。
– 治療方式乃移除食物中的半乳醣(乳糖)
– 易造成白內障
• 形成galactitol (醣醇)的堆積
44
16.2 The Glycolytic Pathway Is
Tightly Controlled
• In metabolic pathways, enzymes catalyzing essentially
irreversible reactions are potential site of control
– Phsophofructokinase, Hexokinase and Pryuvate kinase
• The control of glycolysis in two tissues – skeletal
muscle and liver
– The primary control of muscle glycolysis is the energy
charge of the cell – The ratio of ATP to AMP
– The regulation of glycolysis in the liver illustrates the
biochemical versatility of the liver
45
The control of Muscle glycolysis
• Phsophofructokinase
– The most important control site in the mammalian
glycolytic pathway
– High levels of ATP allosterically inhibit the enzyme
• ATP 會binding 至regulatory site, 而降低酵素與fructose6-phosphate的affinity
• ATP/AMP ratio lower  enzyme activity increase
– Decrease in pH  inhibit enzyme activity
• 肌肉在缺氧狀態下會產生過多乳酸，致使pH值下降，因
此抑制糖解作用，以避免肌肉累積過多的酸。
Fig 16.16 structure of phosphofructokinase
Fig 16.17 allosteric regulation 46
of
phosphofructokinase
Alanine
Fig 16.18 Regulation of glycolysis in muscle
47
The regulation of glycolysis in the liver
•Liver has more-diverse biochemical functions than muscle
–Maintain blood-glucose levels
•Stores glucose as glycogen, and releases glucose
–Use glucose to generate reducing power for biosynthesis
•Phosphofructokinase
–ATP regulation
–Low pH is not a metabolic signal for liver enzyme
–Inhibited by citrate (TCA cycle intermediate)
•High level citrate in the cytoplasm  biosynthesis precusor 
•Enhancing the inhibitory effect of ATP
–Fructose 2,6-bisphosphate as a potent activator
48
血糖濃度增加
fructose-6-phosphate 增加
加速fructose-2,6-phosphate合成
增加phosphofructokinase affinity
Feedforward stimulation
Fructose 2,6-bisphosphate increase
the affinity of phosphofructokinase
and diminishes the inhibitory of ATP
Fig 16.19 Regulation of
phosphofructokinase by
fructose 2,6-bisphosphate
Fig 16.20 Activation of
phosphofructokinase by
fructose 2,6-bisphosphate
•[ATP] increase  allosteric inhibitor
•The inhibitory effect of ATP is reversed
by F-2,6-BP
49
Hexokinase
• The hexokinase reaction in the liver is controlled as in the
muscle
• Another isozyme of hexokinase -- Glucokinase, is not
inhibited by glucose 6-phsophate
– Phosphorylates glucose only when the glucose is abundant
(the affinity for glucose is about 50-fold lower than
hexokinase)
– Provide glucose 6-phosphate for the synthesis of glycogen
and for the formation of fatty acid
• In pancreas β cells, when blood-glucose levels are
elevated
– glucokinase increased formation of glucose 6-phosphate
– Leads to the secretion of the insulin
• Remove glucose from the bloods for storage at glycogen or
50
conversion into fat
Pyruvate kinase (a tetramer of 57kd subunits)
• L type pryuvate kinase predominates in the liver, M type in
muscle and the brain – have many properties in common
L type pyruvete kinase
The glucagon昇糖素triggered cyclic AMP cascade
Phosphorylation of
pyruvate kinase
Diminish activity
由賀爾蒙誘使的磷酸化，
使肝臟不再消耗可提供腦及
肌肉之葡萄糖
Fig 16.21 control of the catalytic activity of pyruvate kinase
51
昇糖素
cAMP-dependent
protein kinase
Protein phosphatase
Regulation of pyruvate kinase
52
A family of transporters enables glucose to enter
and leave animal cells
• Glucose transporters – GLUT1 to GLUT5
– 500 residues of a single polypeptide chain
– 12-transmembrane-helix structure
Normal serum-glucose level: 4mM to 8mM
當血糖過多時，才會將glucose送入
53
Mg2+
Mg2+
2 molecules
Fig 16.2 Stages of glycolysis
54
glyceraldehyde 3-phosphate
Mg2+
Mg2+
Mg2+
K+
Mg2+
K+
Fig 16.2 Stages of glycolysis
55
16.3 Glucose can be synthesized from
noncarbohydrate precusor
• Gluconeogenesis : the synthesis of glucose from
noncarbohydrate precusor
– Especially important during a longer period of fasting
or starvation
– Noncarbonhydrate precusor converted into pyruvate
and then synthesis of glucose
– Major site is the liver (small amount in the kidney, little
in the brain, skeletal muscle or heart muscle)
56
57
Fig 16.24 Pathway of gluconeogenesis
58
Fig 16.24 Pathway of gluconeogenesis
• The major noncarbohydrate precusors for
gluconeogenesis are:
–Lactate
•Lactate is formed by active skeletal muscle
•Readily converted into pyruvate by the action of lactate
dehydrogenase
–amino acids
•Derived from proteins in the diet and during starvation, from
the breakdown of proteins in skeletal muscle
–Glycerol
•The hydrolysis of triacylglycerols in fat cells
59
Gluconeogenesis is not a reversal of glycolysis
Glycolysis
Glucose +ATP
hexokinase
Glucose 6-phosphate + H2O
glucose 6phosphatase
glucose + Pi
glucose-6-phsophate + ADP
ΔG=-33kJ/mol
Froctose 1,6bisphosphatase
Fructose-6-phosphate +ATP phosphofructokinase
flucose-1,6-bisphsophate + ADP
ΔG=-22kJ/mol
phosphoenolpyruvate +ADP
Pyruvate kinase
pyruvate + ATP
ΔG=-17kJ/mol
Fructose 1,6-bisphosphate + H2O
Fructose 1,6-bisphosphate + Pi
Phosphoenolpyruvate
carboxykinase
Oxaloacetate + GTP
Phosphoenolpyruvate + GDP+ CO2
pyruvate + CO2+ ATP + H2O Pyruvate carboxylase
Oxaloacetate +ADP+Pi+2H+
Gluconeogenesis
60
The conversion of pyruvate into phosphoenolpyruvate
begins with the formation of oxaloacetate
Carboxylation
Pyruvate
carboxylase
Inside the mitochondria
Decarboxylation &
phosphorylation
phosphoenolpyruvate
carboxylase
Cytoplasm
Pyruvate + ATP + GTP +H2O  phosphoenolpyruvate + ADP +GDP+ Pi +2H+
61
Pyruvate carboxylase
ATP activation domain
Biotin covalently attached,
serves as a carrier of
activated CO2
Fig 16.25 Domain structure of pyruvate carboxylase
ε-amino group
(amide bond)
N1 atom
Carboxybiotin-enzyme
intermediate
Long, flexible chain
Biotin is not carboxylated
unless acetyl Co-A is bound
一般而言, CO2在身體中通常以HCO3-形式存在
• The carboxylation of pyruvate take place in three stage
– HCO3- + ATP  HOCO2-PO32- + ADP
– Biotin-Enzyme + HOCO2-PO32-  CO2-biotin-enzyme + Pi
– CO2-biotin-enzyme + pyruvate  biotin-enzyme + oxaloacetate
62
Oxaloacetate is shuttled into the cytoplasm and
converted into phosphoenolpyruvate
•Oxaloacetate must be transported to the
cytoplasm to complete the pathway
–Oxaloacetate is reduced to malate
•by NADH-linked malate dehydrogenase in the
mitochondria
–Malate transported across the mitochondrial
–Reoxidized to oxaloacetate
•by NAD+-linked malate dehydrogenase in the
cytoplasm
phosphoenolpyruvate
carboxylase
63
The generation of free glucose is an important
control point
•In most tissues, gluconeogenesis ends in glucose 6-phosphate
– Could form glycogen
– Not transported out of the cell
•The final step in the generation of glucose does not in the
cytoplasm
– Glucose 6-phosphate is transported into the lumen of the
endoplasmic reticulum
– Hydrolyzed to glucose by glucose 6-phosphatase
•Bound to the membrane
•Ca2+-binding stabilizing protein is essential for enzyme activity
– Glucose and Pi are shuttled back to the cytoplasm by transporters
Ca2+-binding stabilizing protein
64
Fig 16.29 Generation of glucose from glucose 6-phosphate
The stoichiometry of gluconeogenesis:
2 pyruvate + 4ATP + 2GTP + 2NADH + 6H2O 
glucose + 4ADP + 2GDP + 6Pi + 2NAD+ +2H+
ΔGo’=-48kJ/mol
65
16.4 Gluconeogenesis and Glycolysis Are
Reciprocally Regulated
Glycolysis is turned on and
gluconeogenesis is inhibited
66
Fig 16.30 Reciprocal regulation of gluconeogenesis and glycolysis in the liver
The balance between glycolysis and
gluconeogenesis in the liver is sensitive to bloodglucose concentration
• In the liver, rates of glycolysis and gluconeogenesis
are adjusted to maintain blood-glucose levels
– signal molecule fructose 2,6-bisphosphate
• stimulates phosphofructokinase
• inhibits fructose 1,6-bisphosphatase
– fructose 2,6-bisphosphate is regulated by
• Bifunctional enzyme (a single 55kd polypeptide chain)
– Phosphofructokinase 2 (PFK2)
» fructose 2,6-bisphosphate is formed
– Fructose bisphophatase 2 (FBPase 2)
» The hydrolysis of fructose 2,6-bisphosphate
» fructose 6-phosphate is formed
67
Glucagon triggers a cyclic AMP signal cascade and activated PKA
(also inactive s pyruvate kinase in the liver)
胰高血糖激素
Insulin stimulates the expression of
phosphofructokinase, pyruvate kinase
and bifunctional enzyme
Fig 16.32 Control of the synthesis and degradation of
fructose 2,6-bisphosphate
68
Substrate cycles amplify metabolic signals and
produce heat
• Substrate cycle such as:
Glycolysis
Gluconeogenesis
– Both reactions are not simultaneously fully active in
most cells
– But some fructose 6-phosphate is phosphorylated to
fructose 1,6-bisphosphate during gluconeogenesis
– Substrate cycles amplify metabolic signals
– Rapid hydrolysis
Some times called futile cycle
69
Lactate:
Fig 16.35 The cori cycle
1. Lactate can be reverted back to pyruvate and metabolized
through TCA cycle
2. The cori cycle
Alanine : like lactate, a major precusor of glucose in liver
• Generated in muscle
• Maintain nitrogen balance
70
71