Download THE CITRIC ACID CYCLE

THE CITRIC ACID
CYCLE
aka Krebs cycle or Tricarboxylic acid (TCA)
cycle
Overview of Citric Acid Cycle
• Feed to cycle is the acetyl group – note that this is already
partially oxidized
• In this cycle, we will complete its oxidation into two
molecules of CO2.
• The electrons lost by carbon will be captured by NAD+
and FAD and fed to oxidative phosphorylation
Overview of Cellular Respiration
The Citric Acid Cycle
Start here
Stage 1 – Oxidize 2-C Units
• Condense acetyl with oxaloacetate
• Animal poison fluoroacetate stops cycle
6-C molecule
4-C molecule
+
Why is Thioester bond so energetic?
-20.9 kJ/mole
-31.5 kJ/mole
Citrate Synthesis Mechanism
Origin of Oxaloacetate
• Can be derived from pyruvate by carboxylation
Pyruvate + CO2 + ATP + H2O
Oxaloacetate + ADP + Pi + 2H+
Citrate Synthesis
• Undesirable side reaction is to hydrolyze the acetyl group
off of acetyl CoA before the acetyl is attached to the
oxaloacetate
• Catalyzed by Citrate Synthase
Open form
Closed form
Citrate Synthesis
• Oxaloacetate binds first and creates binding sites for
•
•
•
•
acetyl CoA – so oxaloacetate is immediately ready for
reaction
The oxaloacetate then reacts with acetyl CoA forming the
unstable compound, citryl CoA
The formation of citryl CoA causes the enzyme to
completely close and brings enzyme residues in close
contact so that water can hydrolyze off the CoA
After desorbing CoA and citrate, the enzyme returns to its
open position
Thus, induced fit minimizes side reactions such as
hydrolysis of acetyl CoA
Citrate Isomerized to Isocitrate
• The isomerization is accomplished by dehydrating the citrate then
hydrating the intermediate, cis-Aconitate
• Why the reaction: easier to oxidize a 2o ROH than a 3o ROH (next
step)
• At equilibrium molecules in this reaction mainly exist as citrate
• However, reaction is pulled to right as the exergonic oxidation
following this step depletes the reaction below of isocitrate thus
shifting it to the right
2o ROH
3o ROH
At equilib:
90%
4%
6%
Alcohols – 1o, 2o, 3o (Reminder)
Citrate to Isocitrate mechanism
Isocitrate is Oxidized and DeCarboxylated (1st Redox)
Isocitrate + NAD+
-ketogluterate + CO2 + NADH
Carboxylate Lewis resonance structures
Detailed chemistry
Second Oxidative De-carboxylation
• Similar to the formation of acetyl CoA by de-carboxylating
pyruvate, we have the same tri-enzyme type of complex
for -ketoglutarate (both an -ketoacid)
-ketoglutarate + CoA + NAD+
Succinyl CoA + CO2 + NADH
-ketogluterate
dehydrogenase
complex
Similar to Pyruvate Conversion to Acetyl
CoA
NAD+
NADH + H+
R
R = CH2-CH2-COO-  ketoglutarate and succinyl CoA
R = CH3 for pyruvate and acetyl CoA
Detailed Chemistry
Analogous to TPP
:B
:B
Flexible amide arm
With disulfide bridge
H:B+
:B
:B
B:H+
Uses FAD to reduce back
to sulfide and NAD+ to oxidize
back to FAD
Stage 1 - 4 Steps to oxidize C to CO2
• So far we’ve had a 2-C moiety enter (acetyl) and we’ve
converted that into 2 moles of CO2
• Generated 2 moles of NADH (high transfer potential
electron carrier)
Stage 2 – 4 Steps produce energy and
regenerate oxaloacetate
• In regenerating oxaloacetate pull more energy out of
cycle.
• In doing so, take 1 step to preserve the thioester bond
energy by phosphorylating GDP to GTP
• Then spend three steps in converting a methylene
group back to a carbonyl carbon in oxaloacetate
Taking Advantage of the Energetic
Thioester Bond
• ΔGo’ for hydrolysis of thioester bond in succinyl CoA is
-33.5 kJ/mole
• Compare to ATP phosphate hydrolysis at -30 kJ/mole
• We preserve that energy by making GTP
• This reaction utilizes a swinging histidine side chain to
transfer the PO42- group from succinyl phosphate to
GDP
Persevering Energy
—Making GTP
;
What is GTP?
• Guanosine Triphosphate (GTP)
• ATP
Regenerating Oxaloacetate-1st Oxidation
• Overall, in order to get back to oxaloacetate need to convert
a methylene group –CH2– by oxidizing to a carbonyl C=O.
• In the first step FAD is reduced rather than using NAD+,
since the free energy change is insufficient to reduce NAD+
• This enzyme is Complex II in the ETC
Succinate
dehydrogenase
Carbon Oxid #
Total Valence e-
-2, -2
46
-1, -1
44
Regenerating Oxaloacetate-1st Oxidation
+ 2H+ + 2e-
FAD + 2H+ + 2e-
FADH2
Detailed Chemistry – Two Views
Succinate Dehydrogenase
• Embedded in the inner
mitochondrial
membrane
• So FADH2 transfers its
electrons directly to
Coenzyme Q (CoQ) in
the Electron Transport
Chain (to be
considered next
chapter).
Regenerating Oxaloacetate-2nd Oxidation
• Fumerate is then hydrolyzed
• Carbon oxid #
-1
-1
0
-2
fumarase
Regenerating Oxaloacetate-3rd Oxidation
• Oxidation of the alcohol carbon gives oxaloacetate
• ΔGo’ = +29.7 kJ/mole (endergonic) ; driven by
consumption of oxaloacetate by converting acetyl
CoA
Maleate
dehydrogenase
Oxidation number
Total valence e-
0
52
+2
50
Oxidation of L-Malate
Energetics in Citric Acid Cycle
Citric Acid Cycle Energetics
• Including Glycolysis all 6 Glucose carbons are
converted to CO2 – 1 in pyruvate to acetyl CoA and two
in Citric Acid Cycle
• And 4 moles of ATP
• Go through cycle twice for each glucose
Citric Acid Productivity
• The 12 reduced species from oxidizing 6 Glucose carbons will
eventually make ATP in the ETC
• 2 NAD+ from Glycolysis
• 2 NAD+ from pyruvate to acetyl CoA
• 6 NAD+ from TCA
• 2 FADH2 from TCA
• Altogether, they will make 34 molecules of ATP
• Together with 4 ATP made in Glycolysis gives theoretical total of 38 ATP
Summary of Citric Acid Cycle
• For each Pyruvate (3-C molecule) go through PDH and
citric acid cycle once or twice/Glucose molecule
• Take in partially oxidized 2-C molecule acetyl
• From this we make in citric acid cycle
• 2 molecules of CO2
• Convert one molecule of GDP to GTP
• Reduce 3 molecules of NAD+ to NADH
• Reduce 1 molecule of FAD to FADH2
• Enzymes in the cycle are closely association with one
another
• Citric acid cycle is anaerobic since no O2 is involved,
however oxygen (ETC) needs to be present to re-oxidize
NADH and FADH2 back to their oxidized forms.
Overall Energy Summary – Theoretical
ATP Yield
Process
ATP/GTP
Reduced Species
ATP
Glycolysis
2
2 NADH
2 x 3 =6
2 NADH
2x3=6
6 NADH
2 FADH2
6 x 3 = 18
2x2=4
Preparation for TCA
TCA
2
Total
4
Total theoretical yield of ATP from glucose is 38 ATP
34
Citric Acid Control
• As we have seen, pyruvate dehydrogenase acts to control
acetyl CoA entering the cycle.
• However acetyl CoA from fat metabolism enters the cycle
directly
• Therefore, the cycle must also have its own means of
control; these are at two points:
• Allosteric enzymes
• Isocitrate dehydrogenase
• -ketogluterate dehydrogenase (like PDH)
Citric Acid Control
• Citric acid cycle has its own control points. Why? Because
other energy sources (fats and proteins) enter metabolism
via acetyl-CoA
• Isocitrate dehydrogenase
• Positively stimulated by ADP which means cell needs more ATP
• Inhibitory are high levels of NADH and ATP
• -ketogluterate dehydrogenase
• Inhibited by its products (succinyl CoA and NADH)
• Why these control points
• When isocitrate dehydrogenase is inhibited, citrate builds up;
citrate then transfers to cytoplasm where it allosterically inhibits
phosphofructokinase to prevent glucose processing
• -ketogluterate accumulation can be used for amino acids and
purine bases
When Energy Needs are Met – TCA used
for Intermediate Generation