TCA Cycle

What happens to pyruvate under aerobic conditions?

the pyruvate is converted to Acetyl-CoA and oxidized to CO2 in the TCA cycle

Where do the electrons go in the TCA cycle?

passed via NADH and FADH2, through the ETC, and then to O2, which is the final electron acceptor

T/F: the TCA cycle produces a lot of ATP

FALSE, the key aspect of the TCA cycle is to pass along electrons that will create a gradient used to power oxidative phosphorylation which will make a lot of ATP

What are the highly regulated steps of the TCA cycle?

1, 3, 4

What enzymes make up the pyruvate dehydrogenase complex?

pyruvate dehydrogenase (e1), dihydrolipoamide acetyltransferase (e2), dihydrolipoamide dehydrogenase (e3)

What does the eukaryotic pyruvate dehydrogenase complex contain?

a unique subunit, termed E3BP which has no known enzymatic function

How does the PDC work?

the active sites of all three enzymes are not far removed from each other, so the product of the first enzyme is passed to the second enzyme without the diffusion of substrates or products- remember this complex is involved in the conversion of pyruvate to acetyl-coa

How large is the eukaryotic PDC?

it is one of the largest-known multienzyme complexes with a diameter of 500 angstroms and a 9.5-megadalton assembly

T/F: glycolysis occurs in the cytoplasm and must find a way to enter the mitochondria in order to get pyruvate to enter the TCA cycle

TRUE

What is the connecting link between glycolysis and the TCA cycle?

the oxidative decarboxylation of pyruvate to acetyl-coa which is catalyzed by pyruvate dehydrogenase

What is the mechanism of the PDC?

1: pyruvate loses co2 and HETPP is formed

2: hydroxyethyl group is transferred to lipoid acid and oxidized to form acetyl dihydroxylipase

3: acetyl group is transferred to coa

What is the core the PDC made of?

e2 subunits, forming either a 24-metric octahedron (gram negative) or a 60-meric pentagonal dodecahedron (in mitochondria of euks and gram positive)

In the mitochondria what does the core structure of the PDC look like?

6 E3BP dimers and 48 E2 subunits- where binding of E3 is mediated by these substituted E3BPs

What do eukaryotic E2 subunits consist of?

an inner catalytic domain and an outer globular domain composed of two lipoid-acid binding sites (L1 and L2) and a small E1-binding domain connected by Ala and Pro rich regions 

What is bound to L1, L2, and L3?

two regulatory enzymes, pyruvate dehydrogenase kinase and a pyruvate dehydrogenase phosphatase

How does the catalytic domain of E2 f(which forms the core of the PDC) catalyze the acetyltransferase reaction?

using acetyl coa as the acetyl donor and dihydroxylipoamide as an acceptor

T/F: there is one lipoic acid residue on each E2 chain

FALSE, there are two

What is the first reaction in the TCA cycle?

the citrate synthase reaction that initiates the cycle! citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-coa

What kind of reaction is the first reaction of the TCA cycle?

a claisen condensation reaction which is a carbon-carbon condensation between a ketone or aldehyde and an ester

What is the mechanism of the first reaction of the TCA cycle?

a nucleophilic attack by the carbanion of acetyl-coa on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis

T/F: the first reaction of the TCA cycle is essentially irreversible

TRUE, although the mitochondrial concentration of oxaloacetate is very low, the strong negative delta g of -31.4 drives the reaction forward- this makes the reaction HIGHLY regulated

What allosterically regulates the first reaction of the TCA cycle?

NADH is an allosteric inhibitor of citrate synthase, as is succinyl-coa formed in the fourth step of the cycle

What is the second reaction of the TCA cycle?

the isomerization of citrate to isocitrate via aconitase

Why is the logical solution isomerization for the second reaction of the TCA cycle?

citrate is a poor candidate for further oxidation because it is a tertiary alcohol which could be oxidized only by breaking a c-c bond-therefore isomerizing the tertiary alcohol to a secondary alcohol allows the cycle to proceed forward; oxidizing a secondary alcohol involves breaking a c-h bond which is way easier than breaking a c-c bond!!

How is aconitase activated?

there is an iron-sulfur cluster in the active site! binding of fe to the vacant position of the cluster is what activates aconitase- this added iron atom coordinates the C-3 carbonyl and hydroxyl groups of citrate and acts as a Lewis acid, accepting an electron pair from the hydroxyl group and making it a better leaving group

Fluoroacetate

an extremely poisonous agent that blocks the TCA cycle in vivo, but has no apparent effect on any of the isolated enzymes. the action of fluoroacetate has been traced to aconitase, which is inhibited in vivo by fluorocitrate which is formed from fluoroacetate in two steps

What is the third reaction of the TCA cycle?

isocitrate is oxidatively decarboxylated to yield a-ketoglutarate which a reduction of NAD+ to NADH in the isocitrate dehydrogenase reaction

How is the aconitase reaction pulled forward?

the -8.34 delta g value of reaction 3 is sufficiently exergonic enough to pull reaction 2 (aconitase) forward!

Why is isocitrate dehydrogenase the first connection of the TCA cycle to the ETC and OP?

because it produces NADH

Why is reaction 3 of the TCA cycle strictly regulated (besides negative delta g)?

because it is a connecting point between two metabolic pathways

How is isocitrate dehydrogenase allosterically activated?

it is activated by citrate and ADP (NADH and ATP are allosteric inhibitors)

What reaction is the first release of CO2 in the TCA cycle?

reaction 3

What is the fourth reaction in the TCA cycle?

a-ketoglutarate dehydrogenase catalyzing the second oxidative decarboxylation producing succinyl-coa, releasing 1 co2 and 1 nadh

What is the mechanism of the fourth reaction of the TCA cycle similar to?

the pyruvate dehydrogenase reaction! this is because a-ketoglutarate dehydrogenase is also a multienzyme complex like the PDC

What is the a-ketoglutarate dehydrogenase complex made of?

a-ketoglutarate dehydrogenase, dihydroxylipoyl transsuccinylase, and dihydrolipoyl dehydrogenase

What is the fifth reaction of the TCA cycle?

succinyl-coa synthetase converting succinyl-coa into succinate

What is unique about succinyl-coa synthetase?

it catalyzes SLP, in which a substrate, rather than an ETC or proton gradient, provides the energy for phosphorylation

What is succinyl-coa used for?

it is a high-energy intermediate that is utilized in the next step of the TCA cycle to drive the phosphorylation of GDP to GTP (in mammals) or ADP to ATP (in plants and bacteria)

Nucleoside Diphosphate Kinase Reaction

a reversible process where an enzyme transfers a phosphate group from a nucleoside triphosphate (like ATP) to a nucleoside diphosphate (like GDP) to create a new nucleoside triphosphate (like GTP); this is how the GTP produced by mammals in the fifth reaction is exchanging its terminal phosphoryl group with ADP

What is the mechanism of succinyl-coa synthetase?

the displacement of coa by phosphate, forming succinyl phosphate at the active site, followed by transfer of the phosphoryl group to an active-site histidine (phosphohistidine intermediate) and release of succinate; the phosphoryl moiety is then transferred from GDP to form GTP

Why is the mechanism of succinyl-coa synthetase important?

it preserves the energy of the thioester bond of succinyl-coa in a series of high-energy intermediates that lead to a molecule of ATP

What is the sequence of steps of how the conversion of succinyl-coa to succinate leads to a molecule of ATP?

thioester → [succinyl-P] → [phosphohistidine] → GTP → ATP

What happens in the first five steps of the TCA cycle?

-a two-carbon acetyl group has been introduced as acetyl-coa and linked to oxaloacetate, and two co2 molecules have been liberated

-the cycle has so far produced two molecules of NADH and one of GTP or ATP and has left a molecule of succinate

How do the last three reactions of the TCA cycle proceed and what is their importance?

the next steps are oxidation steps accompanied by the reduction of an FAD and an NAD+ which provide reducing power in the ETC

What is the 6th reaction in the TCA cycle?

conversion of succinate to fumarate via succinate dehydrogenase

What is unique about succinate dehydrogenase?

It is the only integral membrane protein in the TCA cycle, tightly associated with the inner mitochondrial membrane

What does succinate oxidation involve?

the removal of H atoms across a C-C bond, rather than a C-O or C-N bond, and produces the trans-unsaturated fumarate

T/F: the 6th reaction of the TCA cycle is exergonic enough to reduce NAD+

FALSE, not enough for NAD+, but it does yield enough energy to reduce FAD

Where do the electrons captured by FAD in the 6th reaction go?

they are passed directly into the iron-sulfur clusters of the enzyme and on to coenzyme Q

What is the 7th reaction of the TCA cycle?

fumarase catalyzing the trans-hydration of fumarate to form L-malate

What are the two proposed mechanisms for how reaction 7 of the TCA cycle works?

-it may involve protonation of the double bond to form an intermediate carbonium ion

-there may be an attack by water or oh- to produce a carbanion followed by protonation

What is the 8th reaction of the TCA cycle?

malate dehydrogenase oxidizing malate to oxaloacetate

How is the 8th reaction pulled forward despite a positive delta g?

it is coupled with the favorable citrate synthase reaction

What is meant by steric preferences in NAD+-dependent dehydrogenases?

the enzymes that require NAD+ coenzymes are stereospecific and transfer hydride to either the pro-R or the pro-S positions selectively

Where does the stereospecificity for NAD+-dependent coenzymes arise from?

the fact that the enzymes (especially the active site) are inherently asymmetric structures so the substrate and NAD+ only fit in a certain way

T/F: in order to participate in eukaryotic biosynthetic processes, a-ketoglutarate, succinyl-coa, fumarate and oxaloacetate need to be transported out of the mitochondria

TRUE

Why does the TCA cycle need to be strictly controlled?

because it is situated between glycolysis and the ETC, if unchecked, large amounts of metabolic energy could be wasted in overproduction of reduced coenzymes to ATP or if too slow, not enough ATP would be produced fast enough

Why is the TCA cycle so important?

it is an important source of precursors for biosynthetic processes and must be able to provide them as needed

What are the principal regulatory signals of the TCA cycle?

the concentrations of acetyl-coa, ATP, NAD+, and NADH, with additional effects provided by several other metabolites

What molecule inhibits the main sites of regulation in the TCA cycle?

NADH, so when the cell has produced all the NADH that can be conveniently turned into ATP, the cycle shuts down

Regulation of the TCA cycle by ____, _____, _____, and ____ thus reflects the energy status of the cell

NADH, NAD+, ATP, ADP

What also regulates the mammalian pyruvate dehydrogenase complex?

a Mg2+ dependent pyruvate dehydrogenase kinase; this kinase is activated by NADH and acetyl-coA which stimulates phosphorylation of three Ser residues blocking the first step of the dehydrogenase reaction (decarboxylation of pyruvate)

What allosterically inhibits pyruvate dehydrogenase?

high levels of either acetyl-coa or NADH; acetyl-coa blocks dihydrolipoyl transacetylase (E2) and NADH acts on dihydrolipoyl dehydrogenase (E3)

What does deactivation of pyruvate dehydrogenase do?

it eventually lowers the levels of NADH and acetyl-coA in the matrix of the mitochondrion

How does pyruvate dehydrogenase get reactivated?

it is carried out by pyruvate dehydrogenase phosphatase, a Ca2+-activated enzyme