BMB 401 Citric Acid Cycle

What happens when CAC intermediates are shunted to other pathways?

They must be replenished.

Anaplerotic reactions

Chemical reactions that replenish intermediates.

Step 1 of citric Acid cycle.

this reaction catalyzed by synthase citrate.

Oxaloacetate and Acetyl CoA are condensed to form the intermediate: Citryl CoA.

CoA is the leaving group and the end product is Citrate.

Step 1 reaction type

Condensation

Ordered sequential ( in terms of enzyme kinetics) : Oxaloacetate bind first

Step 1 regulations

NADH (-): High levels, which means no need to make any more of these molecules.

Succinyl- CoA (-): Competitive inhibitor ( competes to bind with Acetyl CoA

Atp (-): High- energy state, which means no need to make any more of these molecules.

Citrate (-): Product inhibition

ALL DECREASE ENZYME ACTIVITY

Is this step reversible or Irreversible

irreversible

What type of protein complex is citrate synthase

Homodimer ( 2 separate proteins that come together)

How does the Acetyl CoA bind to Citrate synthase?

When oxaloacetate is binding, it causes the change in the confirmation of the enzyme to where the binding site for Acetyl CoA is created.

Why must the Acetyl CoA binding site be created?

because of Induced fit. The binding of Oxaloacetate induces the fit of Acetyl-CoA.

What is the purpose of induced fit of Acetyl- CoA?

If Acetyl-CoA bound first it would result in the premature cleavage of that thioester bond before there was anything to condense it to.

It would be a waste of the molecule and energy trapped in that bond.

What happens when the reaction intermediate Citryl CoA forms?

A conformational change that completes active site formation, enabling the cleavage of thioester bonds.

What happens if citryl CoA intermediate is not formed?

The chemistry can't occur to cleave the bond.

How is citrate and CoA formed?

Once the active site is compete, Citryl CoA is cleaved, removing the CoA.

Step 2 of CAC

this reaction catalyzed by Aconitase.

Citrate (Substrate) is taken then isomerized,

The citrate goes through a dehydration reaction and loses a water to form the intermediate cis- Aconitate, and then a hydration reaction occurs and adds a water back, and flips the position of the hydroxyl and hydrogen and this forms the product:

Isocitrate.

Cofactor needed for enzyme

Fe-S cluster: A prosthetic group bound to the enzyme.

Is this reaction reversible or irreversible

reversible

How is it reversible?

It is not regulated by High ATP ratio.

A lot of product = Equilibrium shifts to the left to make more of the substrate citrate

A lot of citrate and not a lot Isocitrate = Equilibrium lies more the right making more Isocitrate

Binding of citrate to the iron-sulfur complex of Aconitase.

In the active site, several iron molecules are coordinated by cystine residues. Citrate is coordinated with an iron. There is also water or hydroxide (depends on Ph)

THIS CLUSTER IS NECESSARY FOR CATALYSIS

Step 3 of CAC

This reaction is catalyzed by Isocitrate dehydrogenase (oxidation reaction).

The first part of the reaction there's the subtrate isocitrate, and NAD+ (cofactor) goes in and forms NADH + H+ in the process. This forms the intermediate: Oxalosuccinate.

In the second half the carbon dioxide is lost, the decarboxylation portion, to form the product:

a- ketaglutarate.

Reaction type

oxidative decarboxylation

Is this step reversible or irreversible

irreversible and is the rate-limiting step for the CAC

Regulators of this step:

NADH and ATP ( Negative)

ADP (positive) - Increases enzyme activity

Step 4 of CAC

This reaction is catalyzed by a-ketoglutarate dehodrygenaue complex (2nd oxidation reaction)

the substrate, A-ketogluturate, and NAD+ and CoA react together to make product SuccinylCoA, CO2, and NADH.

What Cofactors are in involved in step 4?

TPP, Lipoamide, CoA, NAD+, FAD

What type of reaction is this?

oxidative decarboxylation

What are the regulators of Step 4

NADH

ATP

SuccinylCoA- Product inhibition

Is step 4 reversible or irreversible?

Irreversible, Highly regulated

Gene for a-ketoglutarate dehydrogenase complex

The Gene for these different enzymes occur from a gene duplication event so divergent evolution evolved different purposes for very similar complexes.

The difference of these complexes

E1 and E2 are similar to PDC, the only difference is that a- ketoglutarate is used instead of pyruvate.

E3 is same to PDC; has the same goal of repeating the chemistry.

what is the a-ketoglutarate dehydrogenase complex similar too?

Pyruvate dehydrogenase complex

Step 5 of CAC

This reaction is catalyzed by the Enzyme Succinyl CoA Synthetase

The substrates SuccinylCoA, Pi, and ADP forms the major products succinate and Atp. (CoA is also a product)

What kind of reaction type is step 5?

Substrate-Level phosphorylation

What are the cofactors of Step 5?

NONE!!!!!

Is step 5 irreversible

Reversible! The reverse reaction was discovered first, taking succinate, CoAn and ATP to form Succinyl CoA.

Just depends on which reaction is discovered first!!

What is the first step in reaction mechanism of Succinyl CoA Synthetase?

The succinyl CoA and Inorganic phosphate bind by that Pi attacking the carbonyl Carbon in Succinyl CoA, which kicks the CoA off, forming=

Succinyl Phosphate.

What is the second step in reaction mechanism of Succinyl CoA Synthetase?

Succinyl Phosphate has a high transfer potential, meaning it's happy to donate phosphate group. It donates the phosphate group to histidine in active site and free succinate as a product.

This forms Phosphohistidine

What is the Third and fourth step in reaction mechanism of Succinyl CoA Synthetase?

Step 3: The phosphohistidine will swing over to where ADP is bound

Step 4: Substrate level phosphorylation occurs to form ATP and then Histidine is regenerated to do the whole thing over again.

Why might you see ATP or GTP as a product of step 5?

In mammals there are two isozymic forms of the enzyme, one specific for ADP and one for GDP

When will the ADP isozymic form of the enzyme predominate?

In tissues that perform large amounts of cellular respiration (e.g., skeletal and heart muscle)

When will the GDP isozymic form of the enzyme predominate?

In tissues that perform many anabolic reactions (e.g., liver).

seeing succincyl Coa work in reverse, by taking GTP and Succinate as a substrate, to form succinyl CoA for succinct CoA synthesis, then that succincyl CoA is then used in heme synthesis.

What happens if one NTP accumulates but the other is needed?

It is interconverted by nucleoside diphosphokinase but taking a phosphate off one and putting it on the other.

Ex: Taking a phosphate off a GTP and put it onto ADP forming GDP and ATP. (Can do reverse)

Step 6 of CAC

This reaction is catalyzed by the enzyme succinate dehydrogenase.

FAD is needed to Oxidize the the substrate succinate, so it can gain those electrons, then the products Fumarate and FADH2 are formed.

Cofactors involved in step 6

FAD - For CAC in terms of oxidizing succinate this is only needed

Fe-s cluster- used for something else ( a few cards down)

What is the reaction type of step 6

Oxidation

Is step 6 reversible or irreversible

reversible

but doesn't going from fumurate to succinate defeat the the purpose???

If step 6 is reversible, how are we gonna reset the enzyme so that it can take succinate and make another fumarate?

The succinate dehydrogenase is embedded within the mitochondrial membrane and associated with the electron- transport chain.

How is it associated with the ETC?

The electrons from FADH2 are transferred through ETC:

They are transported to another protein that will bring electrons along the ETC, and then FADH2 is reoxidized to FAD for another round for catalysis.

So how does the Fe-S cluster have anything to do with that ETC for this step?

It facilitates the movement of electrons to this other protein that can carry electrons further along the ETC.

Step 7 of CAC

This reaction is catalyzed by the enzyme Fumarase.

The substrate Fumurate and water is going to form the product L-malate.

What type of reaction is step 7?

Hydration

What makes fumarate so special?

It is highly stereospecific and when it is hydrated the water is added across the double bonds is a specific stereochemistry and only form the L form.

what cofactors are needed in step 7?

NONE

Is step 7 reversible or irreversible?

reversible

Step 8 of CAC

The enzyme that catalyzes this reaction is Malate Dehydrogenase (in mitochondria)

The substrate Malate is oxidized by NAD to form Oxaloacetate (and NADH)

What cofactors are needed for step 8?

NAD+

What type of reaction is step 8?

Oxidation

is step 8 reversible or irreversible

Reversible!!

because oxaloacetate is reduced to malate in the mitochondria, transported to the cytosol, and then reoxidized to oxaloacetate by a cytosolic form of malate dehydrogenase to continue gluconeogenesis.

What is this reaction under standard conditions?

It is extremely endergonic. The only reason ΔG is favorable in cells is because the differences in the substrate and product concentrations. Since Oxaloacetate is condense with acetyl CoA in first step so it's constantly being used up, and since that concentration of OXO is low, the equilibrium will push reaction in "forward direction" to form more oxo.

What happens if there is a build up of oxaloacetate in the mitochondria?

Then reverse of the reaction can occur, and there is a lot of reversible steps in the second half of CAC

What is the net reaction of CAC

1 acetyl CoA ---> 3 CO2 + 3 NADH +1 FADH2 + 1 ATP

What are some regulation of the citric acid cycle?

High ATP/ADP ratio

High NADH/NAD ratio

CAC slows down since it's already high of energy (major regulatory places like PDC is slowed and highly regulated enzymes of CAC in first Half are slowed down )

FAVORS GLUCONEOGENSIS (more active since it's storing glucose because there is already a lot of ATP)

What are some regulation of the citric acid cycle? part 2

Low ATP/ADP ratio

Low NADH/NAD ratio:

will do all things needed to make high energy molecules like ATP and NADH. Glycolysis will occur getting ATP and make pyruvate ---> Acetyl CoA by PDC (since it's activated) ----> make a bunch of oxo.

Enhance activity of highly regulated enzymes in first half of CAC.

So what if there was low concentration of oxaloacetate because of Amino acid anabolism in CAC?

Taking the oxo to do other things causing it to have a low concentration. Malate dehydrogenase is turnt up, even tho reversible, causing more oxo to be form and will increase the activity of CAC

Can also make oxo directly through pyruvate by using pyruvate carboxylase

High acetyl CoA concentration of CAC?

Will be cranking through first step of CAC a lot with citrate synthase and increase concentration of citrate. At some point citrate will start inhibiting citrate synthase.

The CAC will go on and make isocitrate but since there is a build up of citrate, isocitrate will build up as well since it's kind of slow and will build up more citrate as a result.

Since PDC makes Acetyl-CoA, PDC will be inhibited because of those high levels .

DECREASE OF CAC

High ATP/ADP ratio in CAC?

Inhibit citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase complex.

DECREASE ACTIVITY

isocitrate dehydrogenase is blocked with inhibitor

Accumulation of isocitrate and citrate

DECREASE in activity

What are defects in CAC that contribute to the development of cancer?

HIF-1

How is HIF-1 activated?

Through hypoxia (Low oxygen) and mutations in succinate dehydrogenase, fumarate, pyruvate dehydrogenase kinase (PDK) (seen in many cancers)

What does HIF-1 do

Enables blood vessel growth and metabolic adaptation (increase in glycolytic enzymes)

How do mutations in isocitrate dehydrogenase contribute to cancer development?

It results in the synthesis of 2-hydroxyglutarate and that modifies methylation patterns in DNA. These modifications can alter gene expression in DNA (How much mRNA is made, how stable it is, and controlling how accessible these genes are) and promote rapid cell growth.