Download 1 Pyruvate and acetate metabolism (The citric acid cycle) I. Pyruvate

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Pyruvate and acetate metabolism (The citric acid cycle)
I. Pyruvate and acetate metabolism to generate 3 CO2
A. Entrance into mitochondria If glucose metabolism were to stop with the formation of
pyruvate, a total of 2 ATP and 2 NADH (containing 2 pairs of electrons removed from
glucose) would be produced. This is indeed the end of the line for glucose metabolism in
the cytoplasm, but another phase of processing awaits pyruvate in the mitochondria.
Two C-C bonds remain to be broken to form 3 CO2 , and in the process 5 pairs of electrons
have to be transferred from each pyruvate onto NAD+ or FADH2 molecules.
The first task is to get pyruvate into the mitochondria. The outer membrane is no
problem - its pores are large enough that pyruvate can float through. The inner, often
corrugated, membrane is not porous to pyruvate, but pyruvate is transported in by a
specific carrier protein in the inner membrane.
Once pyruvate has arrived inside the mitochondria, breaking its C-C bonds proves to be a
complicated task. At first it might appear that the carbonyl on C2 would serve the same
function of weakening the C1-C2 bond as discussed before, but unfortunately its location
on, rather than next to, the C involved in bond-breaking makes it too close to be of use.
Instead, a series of steps involving the pyruvate dehydrogenase enzyme complex and
multiple cofactors is required.
B. Pyruvate Metabolism
1. Decarboxylation via TPP - see V&V p. 543 for mechanism as written on board
The first C-C bond is broken by a set of reactions catalyzed by pyruvate dehydrogenase and
is called a decarboxylation because CO2 is released. (A similar reaction is catalyzed in yeast
by pyruvate decarboxylase which leads ultimately to alcohol production.) A key feature of
the mechanism is the involvement of an enzyme-bound cofactor called thiamine
pyrophosphate (TPP), the structure of which is shown in V&V p. 466 Fig. 16-27. The
thiazolium ring in the center of the molecule directly interacts with pyruvate through its
unusually acidic C (between S and N) which loses its H and becomes a good nucleophile
to attack C2 of pyruvate. (For comparison, the pK of a methyl group is 50, that of a methyl
group adjacent to a carbonyl is 20, and that of the thiazolium C is 12.) As shown in V&V
p. 543, the double bond between C and N of the thiazolium ring is analogous to the Schiff
base seen in the aldolase reaction, and the N likewise serves as an electron sink. After
CO2 is released, the remaining piece of pyruvate stays covalently attached to TPP in a
compound called hydroxyethyl TPP. C2 has a lone pair of electrons, and in yeast, C2
would next be protonated to yield acetaldehyde and then reduced to alcohol, but in other
organisms the following occurs:
2. Oxidation via lipoic acid - see V&V p. 544 for mechanism Hydroxyethyl TPP goes on
to form acetylCoA via another cofactor, lipoic acid (structure in V&V p. 544 Fig. 19-7).
The disulfide in lipoic acid serves as an electron acceptor for the lone pair of electrons on
C2 of hydroxyethyl TPP, resulting in a covalent intermediate and reduction of the
disulfide (step 2). In a subsequent step TPP is released, leaving a high energy thioester
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complex (acetyl dihydrolipoamide in V&V). The nucleophilic S of CoASH then adds to
C2, forming a new C-S bond which replaces that between the acetyl group and lipoic acid
and thus preserves the high energy of the thioester bond (Step 3). Lipoic acid is
subsequently reoxidized by enzyme -bound FAD, which in turn is reoxidized by NAD+ ,
leading to the regeneration of lipoic acid as well as to the production of NADH (step 4 &
5).
C. Acetate metabolism - The citric acid cycle
1. AcetylCoA ----> isocitrate - see text in V&V p. 550 for mechanism
At this stage, one C-C bond remains to be broken, but ironically, now the compound is
too small to have handles for enzymes to work with and the chemistry involved would
be quite difficult. Therefore a new C-C bond must be formed temporarily. This is done
by forming a covalent complex with oxaloacetate via a nucleophilic addition of
acetylCoA’s methyl C to the carbonyl C of oxaloacetate. The resulting compound, citrate,
has an OH group with the potential to be oxidized to a carbonyl, which could in turn
weaken an adjacent C-C bond for cleavage (Fig. 19-12). However, oxidation is impossible
as long as the OH resides on a C with no H’s, so the OH must be transferred to an adjacent
C. The iron-bound enzyme aconitase catalyzes this isomerization, resulting in isocitrate
(Fig. 19-13).
2. Isocitrate ----> succinate ----> oxaloacetate - V&V p. 552-3
Once the OH is on a C with an H, oxidation takes place, resulting in the formation of
NADH and an intermediate called oxalosuccinate. With the carbonyl appropriately
positioned to weaken the adjacent C-C bond, decarboxylation occurs and CO2 is released,
leaving α-ketoglutaric acid (Fig. 19-14). One C-C bonds remain to be broken, but the
compound has a structure similar to pyruvate and can be broken down by the same
mechanism as the conversion of pyruvate to acetylCoA by an analogous three enzyme
complex called α-ketoglutarate dehydrogenase. Thus, α-ketoglutarate undergoes
oxidative decarboxylation, losing CO2 and reducing another NAD+, to form succinylCoA.
Like acetylCoA, succinylCoA has a high energy thioester bond, and in the next step this
energy is used to form GTP from GDP + Pi. At the same time the CoASH group is
released, leaving succinate. Finally, one molecule of FAD one of NAD+ are reduced to
FADH 2 and NADH in the oxidation of succinate back to oxaloacetate over three steps
(Fig. 19-15).
D. Tally
At this point, all of the C-C bonds in the original glucose have been broken to give rise to
CO2. In addition, 2 ATP and 2 NADH have been formed in the cytoplasm, and an
additional 2 GTP and 8 NADH and 2 FADH2 have been formed in the mitochondria
(remember that each glucose gives rise to 2 pyruvate, so everything discussed above is
multiplied by 2).