Lecture 22
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New HW assignment
Anaerobic metabolism (continued)
Other sugars
Gluconeogenesis
Regulation
Alcoholic fermentation (yeast don't have Lactate DH)
O
CO2
C-OCH
3
C=O
CH3
Pyruvate
H-C=O
1. Pyruvate
decarboxylase Acetaldehyde
(TPP) Mg2+, thiamine
NADH, H+
pyrophosphate
NAD+
2. alchohol
dehydrogenase
CH3
H-C-O- H
H
Ethanol
Page 604
Figure 17-26 Thiamine
pyrophosphate (TPP).
Involved in both oxidative and non-oxidative
decarboxylation as a carrier of "active" aldehydes.
Mechanism of Pyruvate Decarboxylase using TPP
1.
Nucleophilic attack by the dipolar cation (ylid) form of TPP
on the carbonyl carbon of pyruvate to form a covalent
adduct.
2.
Loss of carbon dioxide to generate the carbanion adduct in
which the thiazolium ring of TPP acts as an electron sink.
Protonation of the carbanion
Elimination of the TPP ylid to form acetaldehyde and
regenerate the active enzyme.
3.
4.
Page 605
Page 604
Figure 17-25 The two
reactions of alcoholic
fermentation.
Page 606
Figure 17-30 The reaction mechanism of alcohol
dehydrogenase involves direct hydride transfer of the
pro-R hydrogen of NADH to the re face of
acetaldehyde.
Alcoholic fermentation
2ADP + 2 Pi
Glucose
2 Ethanol + 2 CO2
2 ATP
Pyruvate decarboxylase is present in brewer's yeast
but absent in muscle / lactic acid bacteria
Other types of fermentations also exist…
Mixed acid: (2 lactate + acetate + ethanol)
so, in addition to lactate production…
ADP
CoASH
pyruvate
acetyl-CoA + acetyl-P
ATP
NADH, H+
NAD+
lactate
acetaldehyde
NADH, H+
NAD+
ethanol
acetate
Butanediol fermentation
O
CO2 O
C-OCH C-O3
C=O
C-C-O- H
CH3
CH3
2 Pyruvate
O
Acetolactic acid
CO2
CH3
HC-OH
C=O
CH3
Acetoin
NADH, H+
NAD+
CH3
HC-OH
HC-OH
CH3
2,3-butanediol
Other fermentations (Clostridium)
O
CH3-C-COOH
CoA
H2
CO2
CoA
O
isopropanol
OH
CH3-C-CH3
Acetic acid
CH3-C-CoA
Acetyl-CoA
CoA
NADH
O
CoA
CH3-C-CH3
acetone
O
O
CH3-C-CH2-C-CoA
CO2
Other fermentations (Clostridium)
O
O
CH3-C-CH2-C-CoA
H 2O
O
CH3-CH=CH-C-CoA
NADH
NAD
O
CH3-CH2CH2-C-OH
butyric acid
H 2O
O
CH3-CH2CH2-C-CoA
2 NADH
2 NAD
CH3-CH2CH2-CH2-OH
butanol
What about other sugars?
Fructose - fruits, table sugar (sucrose).
Galactose - hydrolysis of lactose (milk sugar)
Mannose - from the digestion of polysaccharides and
glycoproteins.
All converted to glycolytic intermediates.
Fructose metabolism
Two pathways: muscle and liver
In muscle, hexokinase also phosphorylates fructose
producing F6P.
Liver uses glucokinase (low levels of hexokinase) to
phosphorylate glucose, so for fructose it uses a different
enzyme set
Fructokinase catalyzes the phosphorylation of fructose by
ATP at C1 to form fructose-1-phosphate.
Type B aldolase (fructose-1-phosphate aldolase) found
in liver cleaves F1P to DHAP and glyceraldehyde.
Glyceraldehyde kinase converts glyceraldehyde to GAP.
Fructose metabolism
Glyceraldehyde can also be converted to glycerol by
alcohol dehydrogenase.
Glycerol is phosphorylated by glycerol kinase to form
glycerol-3-phosphate.
Glycerol-3-phosphate is oxidized to DHAP by glycerol
phosphate dehydrogenase.
DHAP is converted to GAP by TIM
Page 619
Figure 8.16c Important disaccharides formed by linking
monosaccharides with O-glycosidic bonds.
Lactose, milk sugar.
Galactose metabolism
Galactose is half the sugar in lactose.
Galactose and glucose are epimers (differ at C4)
Involves epimerization reaction after the conversion of
galactose to the uridine diphosphate (UDP) derivative.
1. Galactose is phosphorylated at C1 by ATP
(galactokinase)
2. Galactose-1-phosphate uridylyltransferase transfers
UDP-glucose’s uridylyl group to galactose-1phosphate to make glucose-1-phosphate (G1P) and
UDP-galactose.
3. UDP-galactose-4-epimerase converts UDP-galactose
back to UDP glucose.
4. G1P is converted to G6P by phosphoglucomutase.
Page 621
Mannose metabolism
Mannose is found in glycoproteins
Epimer of glucose at the C2 position
Converted to F6P by two-step pathway
1. Hexokinase converts mannose to mannose-6phosphate
2. Phosphomannose isomerase converts the aldose to
ketose F6P. (the mechanism is similar to
phosphoglucose isomerase with an enediolate
intermediate).
Page 621
Figure 17-37 Metabolism of
mannose.
Entner-Doudoroff pathway
Although glycolysis is nearly universal, some bacteria use
an alternate route called the Entner-Doudoroff
pathway.
Final product is ethanol.
Page 625
Figure 17-38 Entner–Doudoroff pathway for glucose
breakdown.
Sugar catabolic pathways
Glycolysis
Lactate fermentation
Alcohol fermentation
Fructose metabolism
Galactose metabolism
Mannose metabolism
Entner-Doudoroff pathway
Gluconeogenesis
Gluconeogenesis-production of glucose under starvation
conditions since some cells (brain and red blood cells) can
only use glucose as a carbon source.
Noncarbohydrate precursors (lactate, pyruvate, citric acid cycle
intermediates, and carbon skeletons of most amino acids)
can be converted to glucose.
Must go through oxaloacetate (OAA) first.
Lysine and leucine cannot be converted to glucose (degrade to
acetyl-CoA)
Fatty acids cannot be converted to glucose precursors in
animals-degraded completely to acetyl-CoA
Plants can convert fatty acids to glucose with the glyoxylate
cycle.
Glycerol can be converted to to glucose via a DHAP
intermediate
-4 kcal
+0.4 kcal
-3.4 kcal
+5.7 kcal
+1.5 kcal
-4.5 kcal
+1.06 kcal
+0.4 kcal
-7.5 kcal
•3 steps (1, 3, 10)
are considered
irreversible due to
energetics and
inhibitors
preventing the back
reaction.
•Purpose of
gluconeogenesis is
to supply free
glucose for use by
brain or storage
during energy
excess.
•Generally done in
the liver.
Gluconeogenesis (new glucose formation)
•
•
Mainly occurs in the liver.
Shares 7 reversible steps with glycolysis-but must have a mechanism
around the irreversible steps (all Gº’ must be negative).
Step 1
PEP
Pyruvate kinase
ADP
Pyruvate
Gº’= -7.5
ATP
Overcome by circuitous route…
Pyruvate
Pyruvate carboxylase
CO2 ADP
ATP
biotin
OAA
PEP carboxykinase
GTP
PEP
GDP
CO2
Gº’= +0.2
Pyruvate is converted to OAA before PEP
Pyruvate carboxylase catalyzes the ATP driven formation of
oxaloacetate from pyruvate and bicarbonate.
PEP carboxykinase (PEPCK) converts oxaloacetate to
PEP in a reaction that uses GTP as a phosphorylating
agent.
Pyruvate carboxylase
Has a biotin prosthetic group
Biotin enzymes often used for carboxylations with
bicarbonate by forming a carboxyl substituent at its ureido
group.
Page 602
Biotin is an essential human nutrient.
Binds tightly to avidin and streptavidin (can be used as a
linking agent in biotechnological applications b/c of high
affinity).
Page 845
Figure 23-3a Biotin and carboxybiotinyl–enzyme. (a) Biotin
consists of an imidazoline ring that is cis-fused to a
tetrahydrothiophene ring bearing a valerate side chain.
Page 845
Figure 23-3bBiotin and carboxybiotinyl–enzyme. (b) In
carboxybiotinyl–enzyme, N1 of the biotin ureido group is
the carboxylation site.
Long flexible
chain
Enzyme
Page 846
Figure 23-4 Two-phase reaction mechanism of
pyruvate carboxylase.
Page 846
Figure 23-4 (continued) Two-phase reaction
mechanism of pyruvate carboxylase. Phase II
Pyruvate carboxylase
Regulated by acetyl-CoA. (allosteric activator)
Inactive without bound acetyl-CoA.
Inhibition of the citric acid cycle by high levels of ATP and
NADH causes oxaloacetate to undergo gluconeogenesis.
Pyruvate
Pyruvate carboxylase
CO2 ADP
ATP
biotin
OAA
PEP carboxykinase
GTP
PEP
GDP
CO2
Gº’= +0.2
PEP Carboxykinase
Monomeric 608 aa enzyme.
Catalyzes the GTP driven decarboxylation of OAA to PEP
forming GDP
PEPCK cellular location varies with species
Page 602
In mouse and rat liver it is in the cytosol
In pigeon and rabbit liver it is mitochondrial
In humans and guinea pigs it is in both.
Figure 23-5 The PEPCK mechanism.
Page 847
OAA
Gluconeogenesis requires transport
between the mitochondria and cytosol
Enzymes for converting PEP to glucose are in the cytosol.
Intermediates need to cross barriers in order for
gluconeogenesis.
OAA must leave the mitochondria for conversion to PEP or
PEP formed in the mitochondria must go to the cytosol.
PEP tranported across the membrane by specific proteins.
Oxaloacetate has no specific transport system.
OAA must be convertted to either aspartate or malate
Gluconeogenesis requires transport
between the mitochondria and cytosol
The difference between the 2 routes for OAA involves the
transport of NADH.
Page 602
Malate dehydrogenase requires reducing equivalents to
travel from the mitochondria to the cytosol. (uses
mitochonridal NADH and produces cytosolic NADH).
Aspartate aminotransferase does not use NADH.
Cytosolic NADH required for gluconeogenesis so usually
goes through malate.
Page 847
Hydrolytic reactions bypass PFK and
Hexokinase
Page 602
Instead of generating ATP by reversing the glycolytic
reactions, FBP and G6P are hydrolyzed to release Pi in an
exergonic reaction.
Page 848
Glycolysis
Glucose + 2ADP + 2Pi + 2NAD+
Gluconeogenesis
2 Pyruvate + 4ATP + 2GTP
2NADH + 4H+ + 6H2O
2 Pyruvate + 2ATP
+ 2NADH + 4H+ +
2H2O
Glucose + 4ADP
+2GDP + 6Pi + 2NAD+
Net reaction
2ATP + 2GTP + 4H2O
2ADP + 2GDP + 4Pi
Control Points in
Glycolysis
1st reaction of glycolysis (Gº’ = -4 kcal/mol)
HO
O
5
4

6
1
OH
*

2
HO
3
OH
Hexokinase (HK) I, II, II
Muscle(II), Brain (I)
Glucose
OH
Glucokinase (HK IV)
in liver
ATP Mg2+
ADP Mg2+
-2O
3P-O
6
O
5
4
OH
2
HO
3
OH

1
*

OH
Glucose-6-phosphate
(G6P)
Regulation of Hexokinase
• Glucose-6-phosphate is an allosteric inhibitor of
hexokinase.
• Levels of glucose-6-phosphate increase when
downstream steps are inhibited.
• This coordinates the regulation of hexokinase with
other regulatory enzymes in glycolysis.
• Hexokinase is not necessarily the first regulatory step
inhibited.
Types of regulation
1. Availability of substrate Glucokinase (KM 12 mM) vs.
HK (KM = 0.01 - 0.03 mM)
2. Compartmentalization -Brain vs. Liver vs. Muscle
(type I mitochondrial membrane, type II cytoplasmic)
3. Allosteric regulation - feedback inhibition by G-6-P,
overcome by Pi in type I (Brain/ mitochondrial controlled
by Pi levels)
4. Hormonal regulation. Liver has HK as fetal tissue.
Changes to glucokinase after about 2 weeks. If there is
no dietary carbohydrate, no glucokinase. Must have
both insulin and carbohydrates to induce.
2 places where there is no net reaction
PFK
1. ATP + F-6-P
2. F-1,6-P2
Net: ATP
Mg2+
F-1,6-P2 + ADP
F-phosphatase
F-6-P + Pi
Mg2+
ADP + Pi + heat
Similar reaction occurs with hexokinase and G-6-phosphatase.
Generally regulated so this does not occur (futile cycle).
May function in hibernating animals to generate heat.
Primary regulation - reciprocal with energy charge
Enzyme
+
Hexokinase
PFK
F-6phosphatas
e
Pyruvate
kinase
Pyruvate
carboxylase
G-6-P
Pi, ADP,
AMP, F-6-P,
F-2,6-P2
ATP
ATP, citrate,
NADH
K+, AMP, F2,6-P2
Acetyl-CoA
ATP, acetylCoA, cAMP
AMP, F-2,6P2
Major regulation is through energy charge
ATP
ATP
ADP
Gluconeogenesis
Glycolysis
Same reactions make AMP or ADP (primarily in lipid and
nucleotide metabolism)
Adenylate kinase
AMP + ATP
Energy charge
2 ADP
[ATP] +1/2[ADP]
[AMP] + [ADP] + [ATP]
1.0 = 100% ATP Body generally likes it close to 0.9
0.5 = 100% ADP
0 = 100% AMP
Regulation of PhosphoFructokinase (PFK-1)
• PKF-1 has quaternary structure
• Inhibited by ATP and Citrate
• Activated by AMP and Fructose-2,6bisphosphate
• Regulation related to energy status of cell.
PFK-1 regulation by adenosine
nucleotides
• ATP is substrate and inhibitor. Binds to active site and allosteric
site on PFK. Binding of ATP to allosteric site increase Km for ATP
• AMP and ADP are allosteric activators of PFK.
• AMP relieves inhibition by ATP.
• ADP decreases Km for ATP
• Glucagon (a pancreatic hormone) produced in response to low
blood glucose triggers cAMP signaling pathway that ultimately
results in decreased glycolysis.
Effect of ATP on PFK-1 Activity
Effect of ADP and AMP on PFK-1 Activity
Regulation of PFK by
Fructose-2,6-bisphosphate
• Fructose-2,6-bisphosphate is an allosteric activator of
PFK in eukaryotes, but not prokaryotes
•Formed from fructose-6-phosphate by PFK-2
•Degraded to fructose-6-phosphate by fructose 2,6bisphosphatase.
•In mammals the 2 activities are on the same enzyme
•PFK-2 inhibited by Pi and stimulated by citrate