The Citric Acid Cycle – Part I
Chapter 19 - pages 545-556 Outline• review of glycolysis
• the role of the citric acid cycle in all of metabolism • brief overview of the citric acid cycle
• converting pyruvate into acetyl-CoA: getting into the cycle • the first half of the citric acid cycle
• our last lecture covered the mechanisms of carbohydrate storage and controlling the storage and use of sugars
• now is a good time to review glycolysis and get from consumed glucose to two molecules of pyruvate once again…
• then we can take that pyruvate, turn it into acetyl-CoA, and feed it into the citric acid cycle • as we will see today, and for the next lecture, the citric acid cycle is truly the hub of all
metabolism
• however, for our purposes (turning glucose into energy, ATP) I want you all to keep one thing in mind:
- the citric acid cycle will generate electrons for ATP synthesis - REMEMBER: in metabolism, electrons = energy
- but first, let’s review glycolysis
Glycolysis: in Review
• in stage 1 of glycolysis:
• we phosphorylated glucose, isomerized it to fructose-6-phosphate, phosphorylated that and cleaved it, and then isomerized one of the cleavage products to the other (DHAP to GAP) - and we burned two ATPs and made zero…
• stage 2 is two parallel paths that will convert glyceraldehyde-3- phosphate into pyruvate • in stage 2:
• we oxidized and phosphorylated GAP into 1,3-bisphosphoglycerate;
transferred a phosphate from BPG to ADP making two molecules of ATP; isomerized 3-PG into 2-PG; dehydrated 2-PG into PEP and had PEP transfer its phosphate to ADP making two more ATPs - thus giving us our 2 ATP profit and PYRUVATE
• so great, now we’ve made glucose (a hexose) into two molecules of pyruvate (trioses) - WHY?!
- what do we do with pyruvate?
The Role of the Citric Acid Cycle in Metabolism
• aerobic respiration allows far more energy to be released and captured from glucose than the anaerobic methods we discussed before the break
• but, aerobic respiration requires the citric acid cycle
• eventually, glucose will be completely oxidized to _______________________ and __________ while 30-32 ATPs are made
• but, the citric acid cycle is not only involved in breaking down consumed molecules and
releasing energy (________________)
acids, lipids, etc.
• we ended here… now, let’s continue on…
• everything we’ve discussed so far, with regards to glucose metabolism, has occurred in the
_________________ (_______________) - import, glycolysis, etc.
• the citric acid cycle (as well as the electron transport chain and oxidative phosphorylation – to come after exam #2) occurs in the MITOCHONDRIA
• as I’m sure you all remember from high school biology and 23-101/102, mitochondria have an inner and outer membrane
• the inner most compartment of a mitochonrion is the ______________
- the compartment between the two membranes is called the ____________________________ SPACE
• the inner membrane is much more _______________ than the cellular membrane we discussed earlier in the semester
- virtually NOTHING crosses this membrane without a channel
• all of the chemical reactions that makeup the citric acid cycle occur in the MITOCHONDRIAL
___________________
- except for one (we’ll discuss later)
Some Key Features of the Citric Acid Cycle
• essentially, the citric acid cycle is a bunch of ____________ reactions where pyruvate is
oxidized to CO2 and ____________ while electrons are removed and shuttled to the ELECTRON
_____________________ CHAIN
- there, their energy will be slowly released and ____________________
• first, pyruvate is ____________________ - this results in CO2 and acetyl being made - CO2 is released; acetyl is linked to CoA
• in the citric acid cycle, two more CO2 molecules are removed from acetyl-CoA (decarboxylation
reactions) through __________________
- NAD and/or FAD are _____________________ in these reactions - if they’re reduced, then they gained…..?
- and these ______________ are used in the next process called oxidative phosphorylation • but note…
• pyruvate was/is a three carbon molecule - POOH! there went pyruvate
it’s the ________________________ we were after all along!!! • this all happens in the first 2/3rds of the citric acid cycle
- much of the last third is dedicated to ______________________________ the intermediates
allowing the cycle to be just that…a ____________
• first, acetyl is released from CoA and joins with a molecule called ________________________ - this creates another six-carbon molecule called CITRATE
• citrate is _____________________ and successively oxidized with each oxidation resulting in a CO2 being lost - OXIDATIVE DECARBOXYLATION
• we’re left with α-ketoglutarate (an amino acid precursor)
- α-ketoglutarate is oxidatively decarboxylated to make __________________________ • the rest of the cycle is for cycling
• we also make one, single ________ in the citric acid cycle … yipee
- that __________ is used to phosphorylate ADP to ATP … yipee again
• we don’t reap much immediate benefit from this cycle - that’s to come later
• but one thing is obvious (I hope…)
- we gotta get pyruvate turned into acetyl-CoA • so, let’s do it…
Making Pyruvate into Acetyl-CoA
• pyruvate has its own dedicated transporter to enter the ____________________________ • once inside the mitochondria, pyruvate is acted on by the PYRUVATE
___________________________ COMPLEX (know this enzyme complex!)
• PDC will _________________________ pyruvate to acetyl and link that acetyl with CoA
• remember, this is an _____________________ step - acetyl will link to CoA through a –SH (thiol) group - this will result in a high energy bond
- when this bond is broken later, all of that energy will be released and can be used to drive
other _______________________ reactions forward • this entire process involves multiple enzymes found in the PDC
- it is also exergonic (ΔG=-33.4 kJ/mol) and involves a redox reaction
• there are five enzymes that make up this beautiful complex and converting pyruvate into acetyl-CoA takes five steps
• three of the enzymes perform the actual chemistry of the conversion
- the remaining two are a kinase and phosphatase that are involved in __________________ the
• this conversion is a _______________________ point
• this conversion also involves a ______________________ we have not yet seen – LIPOIC ACID
- lipoic acid is often an ______________ agent involved in reactions that use a _________ group • in step one, pyruvate dehydrogenase decarboxylates pyruvate and links what remains – a
hydroxyethyl group – to the coenzyme TPP - this forms hydroxyethyl TPP (HETPP)
• in step two, dihydrolipoyl _______________________ does what its name suggests - transfers the hydroxyethyl group to dihydrolipoic acid
- this step requires the coenzyme lipoic acid and involves the oxidation of the hydroxyethyl group to an acetyl group
• in step three, CoA-SH attacks the bond between acetyl and lipoic acid - this results in the acetyl group being transferred to CoA
- this reaction is also catalyzed by dihydrolipoyl transacetylase
- we’ve successfully made our products; now we must ___________________ PDC
• in step four, the lipoic acid on dihydrolipoyl transacetylase is __________________ back to its original form by the enzyme dihydrolipoyl dehydrogenase – the coenzyme FAD is reduced to FADH2
- FADH2 is in turn oxidized by NAD+ ____________________________ everyone
• overall, when pyruvate is converted into acetyl-CoA, two __________________ are transferred to NAD+ (NADH + H+)
• these are actually our first pair of _______________ (energy) generated by the citric acid cycle - and we’ve not even in the citric acid cycle yet!
• these electrons will be passed on to the electron ___________________ chain
- eventually reducing oxygen to ____________________ and making ATP
The Beauty of the Pyruvate Dehydrogenase Complex • the PDC is a package deal
- everything you need for converting pyruvate into acetyl-CoA is in one complex
• it contains 24 dihydrolipoyl transacetylases arranged in a cube-like orientation (eight trimers) - 12 dimers of pyruvate dehydrogenase on the edges of the cube
- 6 dimers of dihydrolipoyl dehydrogenase on the faces - this arrangement increases speed/efficiency and regulation
The Citric Acid Cycle
• we’ve made acetyl-CoA, so we’re ready to begin…
• OXALOACETATE is the resident ____________________________ of the citric acid cycle - its levels control the speed of the cycle and it must be regenerated if the cycle is to cycle over
and over again
oxaloacetate making ______________ and regenerating CoA - what do you think this CoA will then be used for…?
- this is a _______________________________ REACTION (a carbon-carbon bond was formed) • first oxaloacetate joins acetyl on CoA forming citryl-CoA
• citryl-CoA is then cleaved by hydrolysis to CoA and ______________________ • this reaction is catalyzed by CITRATE SYNTHASE
• this reaction is _______________________ due to the high energy bond linking acetyl and CoA - that energy was released upon hydrolysis driving the entire reaction forward
- this allows us to save ATP
• step two of the citric acid cycle is the ___________________________ of citrate to ISOCITRATE catalyzed by the enzyme ACONITASE
- this isomerization makes an achiral molecule _____________
• aconitase binds citrate unsymmetrically and removes a _____________ molecule - it then adds a water molecule back, but in a different place
• this assymmetric binding allows aconitase to make all isocitrates chiral in the same exact way
• step three of the citric acid cycle is the oxidative decarboxylation of isocitrate to
α-_______________________ – CO2 is released
• this step is catalyzed by ISOCITRATE DEHYDROGENASE and requires two steps
• first, isocitrate is oxidized to form ______________________
- then ________________________ is decarboxylated to yield carbon dioxide and
α-__________________________
• this is our first redox reaction within the citric acid cycle itself - NAD+ is reduced to NADH
• like all other reduced factors in the citric acid cycle this NADH will continue on to the electron
_______________________ chain and eventually be used to make ATP - the grand goal of all of this!
• also remember, this is all happening in ___________________ for every molecule of glucose used… why…?
• step four of the citric acid cycle is another oxidative decarboxylation • α-ketoglutarate is decarboxylated and combines with CoA to make CO2 and
________________-CoA
• very similar to when we made acetyl-CoA, NAD+ is reduced to NADH and these electrons will move on to the electron transport chain
• this process is catalyzed by the α-_____________________________ dehydrogenase complex and – like making acetyl-CoA from pyruvate – requires multiple steps
- in fact, the α-ketoglutarate dehydrogenase complex is very similar to the PDC (three enzymes, TPP, lipoic acid, etc.)
• these reactions are irreversible _____________ and so the citric acid cycle cannot go in reverse • interestingly, the carbons of these CO2 molecules are not from acetyl itself, but from
oxaloacetate
- the carbons from acetyl are used to regenerate oxaloacetate for the next pass of the cycle • since the two carbons of acetyl are gone and we’re right back to a four-carbon molecule, the
rest of the citric acid cycle is dedicated to simply _________________________ oxaloacetate
Summary
• we reviewed glycolysis and got ourselves back to pyruvate
• this pyruvate enters the ________________ of the mitochondria and is acted on by the
PYRUVATE __________________________________ COMPLEX
- pyruvate dehydrogenase decarboxylates pyruvate and links what remains to the coenzyme TPP
- dihydrolipoyl ________________________ transfers this to lipoic acid
- CoA-SH attacks the bond between acetyl and lipoic acid resulting in the acetyl group being transferred to CoA
- the PDC is then regenerated and NAD+ is reduced to NADH
• in step one of the citric acid cycle, the acetyl group from acetyl-CoA is transferred to
oxaloacetate making __________________
• step two of the TCA cycle is the _____________________ of citrate to ____________________ • step three of the citric acid cycle is the oxidative decarboxylation of isocitrate to
α-_____________________________ – CO2 is released
• step four of the citric acid cycle is another oxidative decarboxylation where
α-____________________ combines with CoA to make _________________-CoA • we reduced three NAD+ to NADH and released three CO2 molecules
