Download Lecture 5 - Fermentation and CHO feeder

Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 3300
III. Metabolism
Glucose Catabolism – Part II
Biochemistry 3300
Slide 1
Metabolic Fates of NADH and Pyruvate
Cartoon:
Fate of pyruvate, the
product of glycolysis.
+O2
-O2
Biochemistry 3300
TCA cycle
Fermentation
Slide 2
Metabolic Fates of NADH and Pyruvate
Pyruvate is a central branch point in
Metabolism.
Aerobic pathway:
Citric acid cycle and then respiration;
- yields far more energy (discussed later)
than glycolysis
- relatively slow (limited by O2 transport)
NADH + O2 → NAD+ + Energy
Pyruvate + O2 → 3 CO2 + Energy
Biochemistry 3300
Slide 3
Metabolic Fates of NADH and Pyruvate
Pyruvate is a central branch point in
Metabolism.
Two anaerobic pathways:
-Pyruvate is converted to lactate via
lactate dehydrogenase (ie. muscle cells)
-Pyruvate is converted to ethanol via
ethanol dehydrogenase (ie. yeast)
Anaerobic pyruvate utilization = Fermentation
Both pathways use the NADH (produced in glycolysis):
Overall: Glucose → 2 lactate + 2 ATP
Biochemistry 3300
Slide 4
Lactate Fermentation
Enzyme = Lactate Dehydrogenase
Pyruvate + NADH + + H+
L-Lactate + NAD+
Regenerates NAD+ from NADH (reducing equivalents) produced in glycolysis.
Essential as NAD+ is required for glycolysis (step 6 -GAPDH)
Lactate fermentation is important in red blood cells, parts of the retina and
in skeletal muscle cells during extreme high activity.
Also important in plants and microbes growing in absence of O 2.
∆G’° = -25.1 kJ/mol
Biochemistry 3300
Slide 5
Lactate Dehydrogenase (LDH)
In mammals two different types of LDH subunits are found:
the M type and the H type.
Five forms of the tetrameric isozymes are possible:
M4, M3H1, M2H2, M1H3, H4
H-type predominates aerobic tissues (ie. heart muscle)
H4 LDH has a low KM for pyruvate and is allosterically inhibited by it.
M-type predominates in tissue subject to anaerobic conditions
(ie. liver and skeletal muscle)
M4 LDH has a low KM for pyruvate and is NOT allosterically inhibited by it.
Biochemistry 3300
Slide 6
Lactate Dehydrogenase (LDH)
NADH
LDH monomer
NADH shown as sticks
Catalytic site circled
Redox reaction involving
electron transfer from
NADH to pyruvate.
Biochemistry 3300
Slide 7
Reaction Mechanism of Lactate
Dehydrogenase
Biochemistry 3300
Slide 8
Pyruvate: Terminal Electron Acceptor
of Lactic Acid Fermentation
Fate of Lactate (from fermentation)
Corey Cycle:
Most lactate is exported from the muscle cell via the blood to the liver
↓
Liver converts lactate (back) to glucose
↓
Glucose is transported from liver cells via the blood to the muscle
(stored as glycogen)
The process of transporting lactate to the liver and its
conversion to glucose takes from hours to days to complete.
Biochemistry 3300
Slide 9
Alcoholic Fermentation
Two enzymes involved: Pyruvate decarboxylase irreversible
Alcohol dehydrogenase reversible
Regenerates NAD+ from NADH (reducing equivalents) produced in glycolysis.
Pathway is active in yeast
Second step is reversible
Ethanol can be further metabolised via oxidation that ultimately
produces acetate and enters fat biosynthesis pathways
Biochemistry 3300
Slide 10
Pyruvate Decarboxylase
(Alcohol Fermentation)
Yeast produces CO2 and ethanol in two consecutive reactions
Decarboxylation of pyruvate to acetaldehyde is catalyzed by
pyruvate decarboxylase (PDC) (not present in animals).
PDC contains a tightly non covalently bound coenzyme:
Thiamin pyrophosphate (TPP)
Catalytically active
Biochemistry 3300
Slide 11
TPP Cofactor (Pyruvate Decarboxylase)
The dipolar carbanion (ylid) is the
active form
Decarboxylation of α-keto acids
builds up negative charge on the
carbonyl carbon.
Transition state is stabilized by
delocalization of the developing
neg. charge into a “electron sink”.
Biochemistry 3300
Slide 12
TPP Cofactor (Pyruvate Decarboxylase)
Thiamine Pryophosphate
(TPP)
TPP of Pyruvate Decarboxylase:
Two views related by 90º rotation
about a vertical axis
Biochemistry 3300
Slide 13
TPP Cofactor (Pyruvate Decarboxylase)
How is TPP deprotonated to
its the ylid form?
1) TTP’s aminopyradine ring (subunit 1) is
deprotonated by Glu51 (subunit 2) of the PDC
dimer.
2) amine of aminopyradine deprotonates
thiazolium ring producing ylid form TPP
Note: PDC is a dimer of dimers.
Note: TPP ylid form circled in red
Biochemistry 3300
Slide 14
TPP Cofactor (Pyruvate Decarboxylase)
Thiamine Pyrophosphate
(TPP)
Glu51
Biochemistry 3300
Slide 15
Thiamine Deficiency
TPP addition to carbonyl groups and its ability to act as an
“electron sink”(electron withdrawl) makes it the coenzyme
most utilized in α-keto acid decarboxylations.
Thiamin (vitamin B1) is not synthesized or stored in significant amounts by vertebrates.
Deficiency in humans results in an ultimately fatal condition known as beriberi.
Biochemistry 3300
Slide 16
Alcoholic Fermentation (step II)
Reduction of acetaldehyde to ethanol and regeneration of NAD +
by alcohol dehydrogenase (ADH)
Each subunit of the tetrameric yeast ADH binds one NADH
and one Zn2+.
Biochemistry 3300
Slide 17
Alcoholic Fermentation Part II
Zn2+ polarises the carbonyl oxygen
of acetaldehyde
Hydride ion is transferred from
NADH to the carbonyl carbon
Reduced intermediate acquires a
proton from the medium to form
ethanol.
Biochemistry 3300
Slide 18
Glycolysis:
Substrates other than glucose
Glycogen / Starch
Dietary Polysaccharides
Maltose (Glu-Glu)
Lactose (Glu-Gal)
Sucrose (Glu-Fru)
Biochemistry 3300
Slide 19
Feeder Pathways for Glycolysis
Glycogen metabolism
Glycogen storage granules
in liver
Enzymes of 'feeder pathways'
are underlined in red
Biochemistry 3300
Slide 20
Phosphorolysis:
glycogen / starch degradation
Glycogen phosphorylase /
Starch phosphorylase
- attack of Pi on the (α1→4)
glycosidic linkage of the last
two glucose residues.
Phosphorolysis generates
G1P which must be converted
to G6P (phosphoglucomutase)
to enter glycolysis
Biochemistry 3300
Slide 21
Phosphorolysis:
glycogen / starch degradation
Phosphorylase
- repetitively breaks (α1→4)
linkages until it reaches
an (α1→6)
- produces glucose-1phosphate
Debranching enzyme
- required to break (α1→6)
linkages
- produces glucose
Biochemistry 3300
Slide 22
Phosphoglucomutase mechanism
Glucose 1-phosphate
has to be converted into
glucose 6-phosphate
to enter glcolysis
Where have we previously
seen this type of mechanism?
Biochemistry 3300
Slide 23
Phosphoglycerate Mutase – Reaction 8
Similar mechanism to
phosphoglycerate mutase
(glycolysis)
- different catalytic residue
Biochemistry 3300
Slide 24
Complication! – The Liver
Glycogen is primarily stored in the liver and is used to maintain blood
glucose levels between meals
But … neither G1P nor G6P can be transported out of liver cells
Require separate pathway (below) to convert G6P to glucose for transport
Biochemistry 3300
Slide 25
Dietary Polysaccharides
Dextrin + n H20 → n D-glucose
Dextrinase
Maltose + H20 → 2 D-glucose
Maltase
Lactose + H20 →
D-galactose + D-glucose
Lactase
Sucrose + H20 →
D-fructose + D-glucose
Sucrase
Di- and polysaccharides are converted to monosaccharides,
then funneled into the glycolytic sequence
Biochemistry 3300
Slide 26
Fructose entry into Glycolysis
Two routes for fructose entry
into glycolysis
- tissue specific
Biochemistry 3300
Slide 27
Fructose entry into Glycolysis
Non-Liver
D-Fructose is phosphorylated by hexokinase
and F6P enters glycolysis:
Mg2+
Fructose + ATP → fructose 6-phosphate + ADP
Liver
D-Fructose phosphorylated by
fructokinase (at C1):
Mg2+
Fructose + ATP → fructose 1-phosphate + ADP
Fructose 1-phosphate is then cleaved to
glyceraldehyde and dihydroxyacetone
phosphate (DHAP) by fructose 1-phosphate
aldolase.
DHAP and glyceraldehyde-3-phosphate
Are both glycolytic intermediates
Biochemistry 3300
Glyceraldehyde is phosphorylated by triose
kinase and ATP to glyceraldehyde-3-phosphate.
Slide 28
Galactose entry into the Glycolysis
Galactose entry into glycolysis is
more complex than for other dietary
sugars
Biochemistry 3300
Slide 29
Galactose conversion to
Glucose-1-phosphate
Metabolism of Galactose involves
three enzymes and a sugar nucleotide.
Glycolysis
C1 carbon is activated
as phosphate ester
Biochemistry 3300
Textbook (3rd Edition) has typo
that is corrected here
Slide 30
Galactose conversion to
glucose-1-phosphate
UDP-glucose + Galactose-1-phosphate
↓
UDP-galactose + Glucose-1-phosphate
Must regenerate UDP-glucose to
continue cycle
→ glycolysis
Activation of C1 phosphate
via formation of phosphate
ester with UDP
Biochemistry 3300
Slide 31
Conversion of
UDP-galactose to UDP-glucose
Textbook (3rd Edition) has typo
that is corrected here
Biochemistry 3300
Slide 32