citric acid cycle Flashcards
what is cellular respiration?
the main way energy is produced. cells consume O2 and produce CO2, providing lots of energy ATP and captures energy stored in lipids and amino acids. used by animas, plants, and many microorganisms
three stages of respiration and what they generate (energy)
acetyl CoA production: generates some ATP, NADH, and FADH2
acetyl CoA oxidation: generates more NADH, FADH2, and one GTP
electron transfer and oxidative phosphorylation: generates tons of ATP
where do glycolysis, CAC, and oxidative phosphorylation take place in the cell?
glycolysis = cytoplasm
CAC = mitochondrial matrix
oxidative phosphorylation = inner mitochondrial membrane
respiration step 1
conversion of pyruvate to acetyl CoA
net reaction: oxidative decarboxylation of pyruvate. first carbons of glucose to be fully oxidized (produces CO2)
enzyme: pyruvate dehydrogenase complex. uses 5 coenzymes (TPP, lipyllysine, FAD, NAD+, and CoA-SH) which ones are prosthetic and which are co-substrates?
reactants: pyruvate
products: CO2, acetyl CoA, NADH
details of pyruvate dehydrogenase complex PDC structure
PDC is a multi enzyme complex with three enzymes: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), dihydrolipoyl dehydrogenase (E3).
Advantages of multi enzyme complex: allows channeling of substrates, minimizing side reactions, and allows regulation of entire complex from activity of one subunit
draw the overall reaction of PDC, remember each subunit and cofactors
slide 17
draw the mech for TPP. what is the purpose of the N? what is the part of TPP that is important for chemistry?
chemistry occurs at the thiazolium ring. the N accepts electron pair and allows decarboxylation. slide 12
what other reaction have we seen before that uses TPP? how is it different in respiration?
Ethanol fermentation uses TPP to convert pyruvate to acetaldehyde. the difference is just the absence of lipoyllysine at the last step.
what is important to know about lipoyllysine structure?
it has a very long linker that allows intermediates to move long distances
what is the function of Coenzyme A? what is the functional part of the molecule?
to accept and carry acetyl groups. the reactive thiol group is what functions in chemical reactions
list the 8 steps in the CAC
- C-C bond formation to make citrate
- Isomerization via dehydration/rehedration
- oxidative decarboxylation to give NADH
- 2nd oxidation decarboxylation to give NADH
- substrate level phosphorylation to give GTP
- dehydrogenation to give reduced FADH2
- hydration
- dehydrogenation to give NADH
CAC step 1
C-C bond formation (only C-C bond formation step) by condensation of acetyl-CoA and oxaloacetate reactants: acetyl CoA, oxaloacetate products: citrate enzyme: citrate synthase uses acid/base catalysis rate limiting step activity depends on [oxaloacetate] spontaneous/irreversible mech slide 24
how does citrate synthase avoid unnecessary hydrolysis of thirster in acetyl-coA?
citrate synthase has an induced fit. upon oxaloacetate binding, it changes from the open conformation (which does not allow acetyl-CoA binding) to the close conformation (which does allow acetyl-CoA binding). so acetyl coA is not hydrolyzed unless oxaloacetate is present
CAC step 2
isomerization by dehydration/rehydration
reactants: citrate
products: isocitrate
enzyme: aconitase
citrate is a poor substrate for oxidation, isocitrate is good for oxidation (tertiary vs secondary alcohol)
addition of H2O is stereospecific to cis-aconitate
nonspontateous: [P] kept low
what part of aconitase catalyzes the reaction?
the iron-sulfur center. the sulfurs come from Cys. the center is sensitive to oxidative stress
how is aconitase stereospecific?
the active site has complementary binding points that make citrate orient the correct way for only R isocitrate to be produced. slide 28
CAC step 3
oxidative decarboxylation
reactants: isocitrate. NADP+
products: a-ketoglutarate, NADPH, CO2
enzyme: isocitrate dehydrogenase
loses C as CO2 (most oxidized C, no more reductions possible) and generates NADPH
spontaneous/irreversable: regulated by product inhibition
CAC step 4
final oxidative decarboxylation
reactants: a-ketoglutarate, CoA-SH, NAD+
products: succinyl-CoA, CO2, NADH
enzyme: a-ketoglutarate dehydrogenase complex
Net full oxidation of all Cs in glucose (after 2 cycles) although carbons lost actually came from oxaloacetate
enzyme complex similar to PDC, only differ in substrate sizes
spontaneous/irreversible: regulated by P inhibition
slide 32-33, 17 for mech
CAC step 5
generation of GTP through thioester reactants: succinyl-CoA, GDP, Pi products: succinate, GTP, CoA-SH enzyme: succinyl-CoA synthetase energy of thioester allows for incorporation of inorganic phosphate. goes through phospho-enzyme (His) intermediate non spontaneous: [P] kept low mech slide 37
CAC step 6
oxidation of an alkane to alkene reactants: succinate, FAD products: fumarate, FADH2 enzyme: succinate dehydrogenase bound to mitochondrial inner membrane as part of ETC complex. FAD is unusually covalently bound so FADH2 must be oxidized to restore (cofactor, not catalytic) near equilibrium, reversible: [P] low mech slide 38
CAC step 7
hydration across a double bond reactants: fumarate products: L-malate enzyme: fumarase stereospecific, addition of water is always trans to form L-malate. slightly reversible: [P] low mech slide 40
CAC step 8
oxidation of alcohol to ketone reactants: L-malate, NAD+ products: oxaloacetate, NADH enzyme: malate dehydrogenase final step regenerates oxaloacetate highly unfavorable/reversible: [oxaloacetate] kept very low mech slide 42
net result of CAC (1 turn)
acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O –> 2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+
net oxidation of two carbons to CO2, equivalent to two C from acetyl-CoA but actually form oxaloacetate.
energy captured by electron transfer to NADH and FADH2 (which generate lots of ATP through ETC) and generates one GTP (=ATP)
what does it mean the CAC intermediates are amphibolic? (same thing as anaplerotic??)
they can be removed and put back into the cycle at any intermediate. cycle just needs to have equal input of carbons as the number of carbons lost.
the 4 C intermediates can be formed by carboxylation of 3 C precursors. slide 48 for examples
how is the CAC regulated?
regulation occurs at highly thermodynamically favorable/irreversible steps (PDH, citrate synthase, IDH, and KDH)
general mechanism for all enzymes: activated by substrate availability (NAD+ and AMP), inhibited by product accumulation (NADH and ATP)
how is pyruvate dehydrogenase complex regulated?
by reversible phosphorylation of E1 where phosphorylation makes it inactive. PDH kinase and PDH phosphorylase are part of the complex. kinase is activated by ATP
high ATP = active kinase phosphorylates PDH = less acetyl-CoA
Low ATP = kinase is less active, phosphorylase removes phosphate from PDH = more acetyl-CoA
how is citrate synthase regulated?
inhibited by succinyl-CoA which is an indicator of flow at a-ketoglutarate branch point. if succinyl-CoA is high, cycle is inhibited early on at citrate synthase
how does isocitrate dehydrogenase regulate steps as far back as PFK in glycolysis?
regulating IDH will control citrate levels. aconitase is reversible so inhibition of IDH leads to accumulation of isocitrate and reverses aconitase, accumulating citrate. the citrate leaves the mitochondria and inhibits PFK-1 in glycolysis
how is the glyoxylate cycle different from the CAC?
it has 2 C-C bond forming steps and results in no GTP and only 1 NADH
bypasses decarboxylation with two enzymes: isocitrate lyase and malate synthase.
why is the glyoxylate cycle used?
in plants and some microorganisms (glyoxysome). it is not energy producing, but it feeds into the CAC. It has a net production of 2 acetyl-CoA –> oxaloacetate which allows for net conversion of acetyl CoA to glucose.
