Lecture 32—BCH 4053—Summer 2000
Slide 1
Regeneration of NAD+
• Two possible ways to regenerate NAD +
Recall our discussion about the
isozymes of lactate dehydrogenase,
where different tissues have
enzymes with different kinetic
properties.
• Aerobic regeneration
• Reoxidation by mitochondrial electron transport chain by
mechanisms shuttling electrons into the mitochondria (Chapter
21)
• Anaerobic regeneration
• Reduction of pyruvate to lactate in muscle by the enzyme
lactate dehydrogenase.
• Decarboxylation of pyruvate to acetaldehyde and reduction of
acetaldehyde to ethanol in yeast by the enzyme alcohol
dehydrogenase.
Slide 2
Lactic Acid Fermentation
Excess accumulation of lactate leads
to cramps and muscle fatigue, so
anaerobic work cannot be carried on
indefinitely.
• Anaerobic conditions in muscle
• Lactate dehydrogenase catalyzes reduction of
pyruvate to lactate, regenerating NAD+ .
• (See Figure 19.30)
• Remember the isozymes of LDH (page 467)
• Overall reaction becomes:
Glucose + 2 ADP + 2 Pi → 2 Lactate + 2 ATP
• Lactate is excreted into the blood and sent to the liver for
further metabolism.
Slide 3
Alcoholic Fermentation
• Anaerobic conditions in yeast
• Alcohol dehydrogenase catalyzes reduction of
acetaldehyde to ethanol, regenerating NAD +.
We will explore the interaction of
pyruvate with the thiamine
pyrophosphate prosthetic group in
the next chapter when we discuss
the enzyme pyruvate
dehydrogenase.
• (See Figure 19.30)
• Acetaldehyde is formed from pyruvate by decarboxylation.
• Pyruvate decarboxylase has thiamine pyrophosphate as a
prosthetic group.
• Overall reaction becomes:
Glucose + 2 ADP + 2 Pi → 2 ethanol + 2 CO 2 + 2 ATP
Lecture 32, page 1
Slide 4
Overall Energetics of Glycolysis
• Three steps are far from equilibrium.
• Hexokinase
• Phosphofructokinase
• Pyruvate kinase
• (See Figure 19.31)
Slide 5
Fate of Glucose Carbon Atoms
• To interpret isotopic tracer experiments, it is
important to understand what happens to
each carbon atom of glucose.
• Practice labeling a carbon of glucose and
tracing the label through the pathway.
Slide 6
Fate of Glucose Carbon Atoms,
con’t.
1
H
1 CH2OP
CHO
2
2C O
3 CH2OH
OH
3
HO
H
O
4
H
OH
5
H
OH
6 CH OH
2
Yields
and
or
H OH
6
4
5 H O
HO
HO
1
H
3
H
4 CH
5 HC OH
6 CH2 OP
2
OH
H
O
3,4 C OH
2,5 C O
1,6 CH3
OH
Lecture 32, page 2
Slide 7
Other Sugars in Glycolysis
• Mannose is phosphorylated and isomerized
to fructose-6-phosphate.
• Fructose is phosphorylated to fructose-1phosphate, which is acted on by a special
aldolase. (See Figure 19.32)
• The regulatory enzyme PFK is bypassed.
• Galactose is slightly more complicated.
Slide 8
Metabolism of Galactose
• Phosphorylation at C-1
• Transfer of UDP from UDP-Glc to form Glc-1-P
and UDP-Gal
• Epimerization of UPD-Gal to UDP-Glc
• See Figure 19.33
• Galactosemia is from a defect in the transferase.
• Rarer forms of the disease involve defects in
galactokinase or the epimerase.
In galactosemia, galactose cannot be
metabolized, and its accumulation
causes cataracts, neurological
disorders, and liver problems.
Prevention of the disease consists of
removing galactose and lactose
from the diet. In adults, another
enzyme for activating galactose-1phosphate with UTP alleviates the
problem.
Slide 9
Metabolism of Glycerol
• Glycerol is formed by hydrolysis of
triglycerides.
• Glycerol kinase forms glycerol-3-phosphate
• Glycerol phosphate dehydrogenase converts
it to dihydroxyacetone phosphate, a
glycolytic intermediate.
• (See Figure 19.36)
Lecture 32, page 3
Slide
10
Chapter 20
The Tricarboxylic Acid Cycle
Slide
11
TCA Cycle
aka Krebs Cycle and Citric Acid Cycle
• Occurs inside mitochondria (the matrix)
• Overall reaction:
Acetyl-CoA + 3 NAD + + CoQ + GDP + Pi
↓
2 CO2 + 3 NADH + CoQH2 + GTP + CoASH
Mitochondria have two membranes:
inner and outer. The outer has pores
and is permeable to many things.
The inner is a permeability barrier
which requires specific transporters
for most compounds to move across
it. The matrix is inside the inner
membrane.
• Bridging reaction required that converts
pyruvate to acetyl-CoA.
Slide
12
Bridging Reaction:
Pyruvate → Acetyl-CoA
The reaction is an oxidative
decarboxylation of an alpha keto
acid.
• Catalyzed by Pyruvate Dehydrogenase
• Overall reaction:
Pyruvate + CoASH + NAD+ → Acetyl -CoA + NADH + CO2
• Two cosubstrate coenzymes:
• Coenzyme A and NAD+
• Three prosthetic group coenzymes:
• thiamine pyrophosphate, lipoic acid, FAD
Lecture 32, page 4
Slide
13
Note the first subunit has the same
name as the overall enzyme.
Pyruvate Dehydrogenase
• Multienzyme complex (See Figure p.646)
• Multiple copies of three enzymes, each with
a prosthetic group:
Pyruvate
Dehydrogenase
Dihydrolipoyl
PDH
Thiamine
Pyrophosphate
TA
Lipoic Acid
Transacetylase
Dihydrolipoyl
Dehydrogenase
24 dimers
24 subunits
(cubic core)
DLD
FAD
12 subunits
Slide
14
Pyruvate Dehydrogenase, con’t.
• Mechanism involves two covalent intermediates
with the enzyme:
• Addition of pyruvate to TPP and loss of CO2 forms
hydroxyethyl TPP.
• (This same intermediate is formed by pyruvate decarboxylase
in yeast alcoholic fermentation).
• Transfer of hydroxyethyl group to lipoic acid to form
acetyl lipoic acid.
• (This is an internal oxidation -reduction: acetaldehyde is
oxidized, lipoic acid is reduced.)
• See mechanisms page 646.
Slide
15
Pyruvate Dehydrogenase, con’t.
• Note the role of lipoic acid as a swinging
arm that can move to bind to three different
active sites. It cycles through three
chemical forms.
• The overall reaction is irreversible.
• The enzyme is found inside the
mitochondria. Therefore pyruvate must
cross the mitochondrial membrane.
Lecture 32, page 5