Download Lecture 6 - TCA cycle I - University of Lethbridge

Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 3300
III. Metabolism
The Citric Acid Cycle
Biochemistry 3300
Slide 1
Cellular Respiration (combustion)
Cellular respiration is the step-wise release of energy from glucose,
fatty acids and (some) amino acids.
•Efficient aerobic process that requires oxygen and produces carbon dioxide.
Energy from these reactions is used to synthesize ATP molecules.
Involves the complete oxidation
of glucose to carbon dioxide
and water.
C atoms* (glucose)
C atom (CO2)
Biochemistry 3300
Oxidation Number
4
0
Slide 2
Catabolism of Proteins, Fats, and
Carbohydrates
Cellular respiration involves
three main phases:
Phase 1
Carbon skeletons of
organic fuel molecules
are degraded to acetyl
groups that are attached
to acetyl-CoA
Biochemistry 3300
Slide 3
Catabolism of Proteins, Fats, and
Carbohydrates
Cellular respiration involves
three main phases:
Phase 1
Carbon skeletons of
organic fuel molecules
are degraded to acetyl
groups that are attached
to acetyl-CoA
Phase 2
Oxidation of acetyl groups
in the citric acid cycle
Biochemistry 3300
Slide 4
Catabolism of Proteins, Fats, and
Carbohydrates
Cellular respiration involves
three main phases:
Phase 1
Carbon skeletons of
organic fuel molecules
are degraded to acetyl
groups that are attached
to acetyl-CoA
Phase 2
Oxidation of acetyl groups
in the citric acid cycle
Phase 3
Electrons carried by NADH
and FADH2 are funneled
into the respiratory chain.
Biochemistry 3300
Slide 5
Catabolism of Proteins, Fats, and
Carbohydrates
The citric acid cycle is also
called the Krebs cycle or
the tricarboxylic acid
(TCA) cycle.
The citric acid cycle is the
“hub” of the metabolic system.
- Majority of carbohydrate, fatty acid
and amino acid oxidation.
- Majority of the generation of these compounds
and others.
Citric Acid Cycle is amphibolic as it acts
both catabolically and anabolically
Biochemistry 3300
Slide 6
History
By 1930, it was established that some compounds:
Carboxylic acids (acetate, lactate)
Dicarboxylic acids (succinate, malate, -ketoglutarate) and
Tricarboxylic acids (citrate, isocitrate)
would stimulate O2 consumption and CO2 production when added to
“minced” muscle
1935: Albert Szent-Gyorgyi
Succinate → Fumarate → Malate → Oxaloacetate
Carl Martius & Franz Knoop
Citrate [→ Cis-aconitate] → Isocitrate → -ketoglutarate →
Succinate → Fumarate → Malate → Oxaloacetate
Biochemistry 3300
Slide 7
History
Subsequently, Martius & Knoop showed:
Pyruvate and Oxaloacetate can form citrate non-enzymatically (requires
peroxide and basic conditions).
Odd: oxaloacetate is the product in their pathway!
And then Hans Krebs the showed:
Succinate is formed from fumarate, malate or oxaloacetate.
Odd: These appear to be the reverse reactions!
Citric Acid metabolic pathway is a CYCLE !!
Biochemistry 3300
Slide 8
Catabolism of Proteins, Fats, and
Carbohydrates
Glycolysis – cytosol
TCA cycle – mitochondria (eucaryotes)
Biochemistry 3300
Slide 9
Citric Acid Cycle enzymes are
in the mitochondrial matrix
Substrates must cross both the outer and inner
mitochondrial membrane
Biochemistry 3300
Slide 10
Mitochondrial
Membrane
Particles visualized by
EM are “large”
protein complexes
Inner membrane is
rich in large protein
complexes
Biochemistry 3300
Slide 11
Coenzyme A
Nathan Kaplan and Fritz Lipmann discovered Coenzyme A (CoA)
Ochoa and Lynen showed that acetyl-CoA is an intermediate in the
conversion of pyruvate to citrate.
Biochemistry 3300
Slide 12
Pyruvate is oxidized to
acetyl-CoA and CO2
Combined dehydrogenation and decarboxylation of pyruvate
requires the sequential action of three different enzymes
(E1, E2, E3) and five different coenzymes.
Biochemistry 3300
Slide 13
Pyruvate Dehydrogenase
Complex (PDH)
PDH complex contains three subunits, present in multiple
copies. Number varies among species.
E. coli
yeast
Pyruvate dehydrogenase
--
E1
24
60
Dihydrolipoyl transacetylase
--
E2
24
60
Dihydrolipoyl dehydrogenase
--
E3
12
12
Molecular weight of 4,600,000 Da ;
50 nm in diameter
Lipoate is connected to E2
Biochemistry 3300
Slide 14
Pyruvate Dehydrogenase Complex
Requires Five Coenzymes
1) Nicotinamide adenine dinucleotide (NAD+)
2) Thiamin pyrophosphate (TPP)
3) Flavin adenine dinucleotide (FAD)
4) Coenzyme A (CoA)
5) Lipoate
The lipoyllysl moiety acts as
a carrier of both hydrogen and
an acetyl group.
Biochemistry 3300
Slide 15
Structure
Cryo-EM reconstruction of PDH from bovine kidney
Biochemistry 3300
Slide 16
Structure
E2 consists of three types of domains linked by short polypeptide linkers.
Biochemistry 3300
Slide 17
Structure and Mechanism
Oxidative decarboxylation of pyruvate to acetyl-CoA.
Step 1 is rate limiting and responsible for
substrate specificity.
Biochemistry 3300
Slide 18
Structure and Mechanism
Decarboxylation of pyruvate and formation
of acetyl lipoyllysine
Biochemistry 3300
Slide 19
Structure and Mechanism
Formation of Acetyl-CoA
Biochemistry 3300
Slide 20
Why such a complex set of
enzymes?
1. Enzymatic reaction rates are limited by diffusion, with shorter
distance between subunits in an enzyme, the substrate can be
directed from one subunit (catalytic site) to another.
2. Channeling metabolic intermediates between successive enzymes
minimizes side reactions. (Substrate channeling).
3. Local substrate concentration is kept high.
4. The reactions of a multienzyme complex can
be coordinately controlled / regulated.
Biochemistry 3300
Slide 21
Arsenic Compounds are
Poisonous
-
O
S
HS
OH
O- As
+
As
OH
S
HS
R
R
As(III) compounds, such as arsenite (AsO33-) and organic arsenicals,
are toxic because they covalently attach to sulfhydryl compounds.
Vicinal (adjacent) sulfhydryls form bidentate adducts (top right)
Biochemistry 3300
Slide 22
Structure and Mechanism
Arsenite
inhibits
E3
Biochemistry 3300
Slide 23
Mechanism of
Dihydrolipoyl Dehydrogenase.
More complicated than expected:
1. Spectra of oxidized dihydrolipoamide dehydrogenase (E3) is
unaffected by arsenite.
2. NADH reaction with the oxidized enzyme in the presence of
arsenite → forms an enzymatically inactive species.
3. Spectrum of the arsenite-inactivated enzyme (2.) indicates that its
FAD prosthetic group is fully oxidized.
Recall: The oxidation state of the flavin in a flavoprotein is readily
established from its characteristic UV-Vis Spectrum:
FAD is intense yellow, whereas FADH2 is pale yellow.
Explanation ?
Biochemistry 3300
Slide 24
Mechanism of
Dihydrolipoyl Dehydrogenase.
Oxidized dehydrolipoamide dehydrogenase has
an additional electron acceptor.
Arsenite inhibition suggests a disulfide as acceptor.
See X-Ray structure of dehydrolipoamide DH
from P. putida, PDBID 1LVL
Catalytic active residues: Cys 43 & 48 , Tyr 181
Biochemistry 3300
Slide 25
Mechanism of
Dihydrolipoyl Dehydrogenase.
Arsenite
target
Biochemistry 3300
Slide 26
Catalytic Cycle
of
Dihydrolipoyl
dehydrogenase
Biochemistry 3300
Slide 27
Eight Steps of the Citric Acid Cycle
NEXT
Biochemistry 3300
Slide 28