Download Chapter Nine - The Krebs Cycle

Introduction to the Krebs Cycle
• Hans Kreb discovered its cyclic nature
• Goes by three names
– Citric acid cycle
– Tricarboxylic cycle
– Krebs cycle
9.2 A Cyclic Pathway
• Linear pathways can also be viewed as cyclic
– Example – ethanol from pyruvate in yeast
•
•
•
•
Pyruvate decarboxylase has several forms
Pyruvate enters cycle
Products CO2 and acetaldehyde leave
Acetaldehyde and NAD then enter and NADH and
ethanol leave
– Every enzymatic reaction can be written as a cycle
• One molecule of an enzyme can catalyze infinitely many
conversions
9.2 Acetyl CoA: Substrate of the Krebs Cycle
• Overall process: oxidation of two acetyl carbons of acetyl-CoA
completely to CO2
– high energy electrons are captured
• Acetyl CoA
– Thioester of acetate with CoA
– Free SH group
• Formation of acetyl – CoA from pyruvate requires enzyme complex
• Occurs in mitochondria
• Cytosolic pyruvate must cross two membrane of mitochondria
– Outer membrane can be crossed using porins
– Inner membrane can be crossed using transport proteins
9.2 Acetyl CoA: Substrate of the Krebs Cycle
• Pyruvate dehydrogenase complex
– Enzyme complexes are groups of enzyme that catalyze a
metabolic sequence without releasing intermediates
– Involves 3 separate enzymes
– Five cofactors
• Mobile cofactors – CoA, NAD and NADH
• Bound cofactors – thiamine pyrophosphate, lipoic acid
and flavin adenine dinucleotide
9.2 Acetyl CoA: Substrate of the Krebs Cycle
• Overall reaction is pyruvate + CoA + NAD+ which
produce Acetyl-CoA and NADH
• Mechanism
– E1 first reacts with
– E2 froms acetyl-thioester and regenerates E1
– Mobile cofactor CoA-SH displaces acetyl group from
E2
– E2 needs to be regenerated
– FAD is regenerated using mobile cofactor NAD
9.3 Overview of Carbon Flow
• Intermediates of Krebs cycle are
six, five and four carbon compounds
• Input to pathway is 2 carbon
acetyl CoA condenses with
4 carbon compound
• First CO2 is lost in the subsequent
reactions - CO2 is from 4 carbon skeleton,
not the incoming acetyl CoA
9.3 Overview of Carbon Flow
• Second CO2 is lost in another reaction
– Again from the carbon skeleton and not the
incoming acetyl CoA
• Resulting 4 carbon intermediate is converted
to a molecule with plane of symmetry
9.4 Steps of the Pathway
• Citrate Synthase reaction
– Introduces new carbon into pathway
– Overall reaction – oxaloacetate + acetyl CoA
produces citrate and CoA
– Metabolically irreversible
– No allosteric modulators
9.4 Steps of the Pathway
• Citrate Synthase reaction cont.
– Mechanism –
• Extraction of a proton from terminal methyl group of
acetyl CoA
• Nucleophilic substitution leads to citryl-CoA
intermediate
• Hydrolyzed to citrate
9.4 Steps of the Pathway
• Aconitase
– Overall reaction – citrate is dehydrate to cisaconitate and then rehydrated to isocitrate
– Mechanism
• Requires Fe-S cluster
» Fe2+ ions play a dual role
• Hydroxyl group attacks carbon 5
» Produces isocitrate
9.4 Steps of the Pathway
• Aconitase cont
– Creates chiral carbon
• Enzyme has a three point landing
• Prochiral center
– Identifies a carbon with two identical substituents
that can be discriminated
9.4 Steps of the Pathway
• Fluoroacetate Poisoning involves the first two
enzymes of the Krebs Cycle
• Fluoroacetate – active ingredient in poisons
used in rodenticide
• Enzymatic conversion to an inhibitor
– citrate synthaste reactions creates fluorocitrate
– Fluorocitrate is irreversible inhibitor of aconitase
9.4 Steps of the Pathway
• Isocitrate dehydrogenase
– Overall reaction – oxidation of isocitrate to
produce 2-ketoglutarate
• NAD is used as a mobile cofactor
• CO2 is also released
– Metabolically irreversible reaction
9.4 Steps of the Pathway
• Isocitrate dehydrogenase cont.
– Mechanism
• Oxidation to chemically unstable beta keto acid
intermediate
• Decarboxylation
• Enol- keto form are in equilibrium
9.4 Steps of the Pathway
• -Ketoglutarate DH Complex
– Overall reaction – 2 ketoglutarate reacts with acetyl CoA
and NAD to produce succinyl CoA, NADH and CO2
– Enzyme complex
– Similar mechanism to pyruvate dehydrogenase complex
– Metabolically irreversible
– Important regulatory site of Krebs cycle
9.4 Steps of the Pathway
• Succinyl – CoA synthetase
– Can also be called succinate thiokinase
– Overall Reaction - Succinyl CoA reacts with GDP and Pi
to form succinate, GTP and CoA-SH
– Only reaction in Krebs cycle to form high energy
phosphate
– Metabolically irreversible reaction
9.4 Steps of the Pathway
• Succinyl CoA synthetase cont.
– Mechanism
• Displacement of thioester by inorganic phosphate
• High energy phosphate is transferred to histidyl group
• High energy phosphate is transferred from the histidyl
group
– GTP can be directly used as energy intermediate
9.4 Steps of the Pathway
• Succinate Dehydrogenase
– Overall reaction – conversion of succinate to
fumarate
– Only membrane in Krebs cycle that is membrane
bound
– Ubiquinone is used as a redox cofactor –
– Metabolically irreversible
• No known regulators
9.4 Steps of the Pathway
• Succinate dehydrogenase cont
– Enzyme complex
• Catalyzes series of electron transfers
• Electrons are removed from the interior C-C bond
• Using ubiquinone as electron carrier
– Also plays a role in oxidative phosphorylation
– Inhibitor – dicarboxylate malonate –
9.4 Steps of the Pathway
• Fumarase
– Overall reaction – hydration of fumarate to malate
– Near equilibrium enzyme
– Mechanism
• Adds water across the C-C double bond
– New chiral center is created
• Has Fe-S complex for substrate positioning
9.4 Steps of the Pathway
• Malate dehydrogenase
– Overall reaction – malate is converted to
oxaloacetate
• NAD is used as a mobile cofactor
– Near equilibrium reaction
– Mechanism
• Transfer of hydride from C-H bond to NAD ring
– Oxaloacetate product is achiral
• Reverse reaction creates a chiral malate
9.5 Energy Balance
• Assumptions can be made as to how much
energy is extracted by energy cofactors
– NADH = 3 ATP
– FADH2 = 2 ATP
– GTP = 1 ATP
• Using these assumptions, we can estimate a total
of 12 ATP per cycle
– 3 NAD linked dehydrogenase reactions produce 9 ATP
– 1 FADH2 reaction creates 2 ATP
– Succinyl CoA synthetase reaction produces 1 ATP
9.6 Regulation
• Two levels of regulation
– Supply of substrate
– Intrinsic activity
9.6 Regulation
• Supply of substrate – Acetyl CoA – can
regulate
– Acetyl CoA can come from carbohydrates, fats and
proteins
– Regulated by phosphorylation and
dephosphorylation of E1 component
• Catalyzed by protein kinases and phosphatase
대사과정의 요약.
9.6 Regulation
• Intrinsic acitivity
– Several steps are metabolically irreversible
•
•
•
•
•
Citrate synthase
Isocitrate dehydrogenase
2-ketoglutarate dehydrogenase
Succinyl CoA synthetase
Succinate dehydrogenase
– All of the above steps are regulated by Calcium
9.6 Regulation
• Citrate levels can affect glycolysis
– Overall increase in flux of Krebs cycle increases
citrate
– Citrate can be transported across inner
mitochondria membrane
– Decreases phosphofructokinase activity and
glycolysis
9.7 Krebs Cycle as a Second Crossroad
of Metabolic Pathways
• Can be viewed as a metabolic hub
• Fatty acid synthesis uses citrate
• Some amino acids can be converted to
intermediates
– Transamination
• Heme biosynthesis uses succinyl CoA
• Glyoxylate cycle in bacteria and plants