bio.lesson document 15

8/6/2019 bio.lesson document 15

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BIO 202 Biochemistry II

bySeyhun YURDUGL

Lecture 5

The Citric Acid(Tricarboxylic

Acid/Krebs) Cycle


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Outline

Introduction

Reactions

Energy balance Regulation and linkage within the

intermediary metabolism


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The Citric Acid Cycle

also called the tricarboxylic acid (TCA) cycle;

and the Krebs cycle.

the final common catabolic pathway for theoxidation of fuel molecules.

Two carbons enter the citric acid cycle as acetyl

CoA; and two carbons leave as CO2.


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The Citric Acid Cycle

In the course of the cycle, four oxidation-reduction reactions take place;

to yield reduction potential in the form ofthree molecules of NADH;

and one molecule of FADH2.

A high energy phosphate bond (GTP) isalso formed.


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Linkage to other metabolic

pathways


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Since cycle intermediates can beincorporated into both anabolic

and catabolic pathways, the cycleis really amphibolic, not justcatabolic


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Anaplerotic pathway

If an intermediate in any pathway: replenished by another pathways

intermediate: A typical example of anaplerotic pathway. E.g.malate in the mitochondrial matrix

replenishes pyruvate.

Anaplerotic: ofGreek origin, meaning to fillup.


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The first reaction:

Pyruvate to Acetyl Co-A In reality, this reaction:

not really in the Krebs Cycle, but since it is the first reaction that occursin the mitochondrion;

and it leads directly into the cycle,

it is usually included in the discussions ofthe cycle.


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The first reaction:

Pyruvate to Acetyl Co-A In this reaction, pyruvate, a three carbonmolecule that is generated in glycolysisand;

in the metabolism of some amino acids,

is decarboxylated (a carboxyl group isremoved) to the two carbon acetate


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The first reaction:

Pyruvate to Acetyl Co-A The carboxyl group: released as carbon dioxide.

catalyzed by the enzyme pyruvatedehydrogenase.

This reaction is also an oxidation;

as 2 electrons are removed from pyruvateduring the reaction


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Pyruvate Dehydrogenase Complex

Structure of the thiamin diphosphate dependent enzyme

pyruvate decarboxylase-brewer's yeastSaccharomyces

cerevisiae uvarum strain


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Pyruvate Dehydrogenase Complex

The lipoyl E2 domain of complex which serves as an acyltransferase.

From: Azotobacter vinelandii


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The first reaction:

Pyruvate to Acetyl Co-A The two electrons:

accepted by NAD and results in theformation of NADH.

This oxidation is very exergonic (G = -7.5kcal/mole).


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The first reaction:

Pyruvate to Acetyl Co-A Some of the energy released from thisreaction: transferred with the electrons toNADH;

and some: used to energize acetate byadding coenzyme A to acetate;

forming acetyl CoA, the actual product ofthe reaction


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Note about this reaction:

Pyruvate; the product of glycolysis, comesfrom glucose.

It may also come from some amino acids. reaction occurs in the mitochondrion.


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One carbon:

removed from pyruvate in the form of

carbon dioxide (note the yellow carbon inthe figure of slide 10).

This leaves just two carbons remainingfrom pyruvate.


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The addition of the coenzyme A to theacetate (see red in the above figure ofslide 8);

acts to conserve the energy;

released from the reaction and to energizethe acetate.


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Step 2: Condensation

Acetyl Co-A to Citrate via

Oxaloacetate

In step 1 of the Krebs cycle,

the two-carbon compound, acetyl-S-CoA,

participates in a condensation reactionwith the four-carbon compound,

oxaloacetate, to produce citrate:


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Oxaloacetate

moderately exergonicreaction.

Thermodynamically, the equilibrium is infavor of the products.

considered to be the first committedstep

of the Krebs cycle


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Acetyl Co-A to Citrate viaOxaloacetate

Being the first committed step,

this is a likely step to have some kind ofregulatory control mechanism.

which will effectively regulate the entire cycle.


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Oxaloacetate

The Krebs cycle is also known as the citric

acid cycle. Citrate is a tricarboxylic acid,

and the Krebs cycle is also known as the

tricarboxylic acid(orTCA) cycle


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In other words: Oxaloacetate +

Acetyl CoA to Citrate

Enzyme: Citrate synthase Reaction: Condensation

Oxaloacetate condenses with acetyl CoA toform citryl CoA.

Then citryl CoA is hydrolyzed to citrate andCoA.

Prosthetic group: No


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Step 3. Isomerization of

Citrate

a decarboxylation reaction.

usually involve - (or -) keto acids hydroxyl group of citrate can be oxidizedto yield a keto group,

but to form an -keto acid;

it needs to be adjacent to one of theterminal carboxyl groups


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Aconitase

Aconitase (E.C.4.2.1.3) in the activated (4Fe-4S)cluster form.


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Step 3. Isomerization of Citrate


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Citrate to cis-Aconitate

Enzyme: Aconitase

Reaction: DehydrationCitrate: isomerized to isocitrate by this first

dehydration;

and yields cis-aconitate asan intermediate.

Prosthetic group: Fe-S


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cis-Aconitate to Isocitrate

This reaction is endergonic, so the equilibrium is in favor of the

reactants;

and not the desired product. However, the exergonic character of the

nextreaction in the cycle:

helps shift the equilibrium ofthis reactiontowards the right.


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cis-Aconitate to Isocitrate

Two asymmetric centers in the D-Isocitratemolecule:

Each can adopt either the L- or D-form, thus there are 4 possible isomers of thismolecule


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Aconitase enzyme

only produces the single form of Isocitrate(D-Isocitrate).

a stereospecificenzyme


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Step 4: Generation of CO2 by

an NAD

+

linked enzyme

The Krebs cycle contains two oxidative

decarboxylation steps; this is the first one

The reaction is catalyzed by the enzymeI

socitrate dehydrogenase


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Isocitrate Dehydrogenase

Isocitrate Dehydrogenase (E.C.1.1.1.42) with NADP

St 5


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Step 5:

alpha-Ketoglutarate to Succinyl

CoA

Enzyme: alpha-Ketoglutarate dehydrogenasecomplex

Reaction: Oxidative decarboxylation almost as same as the reaction of the oxidative

decarboxylation of pyruvate to acetyl CoA; by pyruvate dehydrogenase complex.

reaction gives one NADH. Prosthetic group: Lipoic acid, FAD, TPP


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Step 6: Succinyl CoA to

Succinate

only thisstep givesa high-energyphosphate compound,

GTP from the couple reactions of thethioester bond cleavage

and the phosphorylation of GDP.

Prosthetic group:No


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Succinyl-CoA Synthetase

Succinyl-CoA Synthetase (E.C.6.2.1.5) with coenzymeA


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Succinate to fumarate

succinate dehydrogenase complex:

also known as complex II of the electron

transport system, thus the oxidation of succinate to fumarateis the only Krebs reaction;


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that takes place on the inner membraneitself,

the other reactions: catalyzed by soluble

enzymes. The energy carrier flavin adenine

dinucleotide (FAD):

also a part of the succinatedehydrogenase complex


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as the enzyme and FAD: both part of the same complex, the only step needed to initiate succinate

oxidation: the binding of succinate to the enzyme. mitochondria succinate supported respiration

can usually be accomplished,

as long as fragments of the inner membraneremain


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Malate to Oxaloacetate

Enzyme: Malate dehydrogenase

Reaction: Oxidation

Malate: dehydrogenated to formoxaloacetate.

The hydrogen acceptor: NAD+.

S

o this reaction yields NADH. Prosthetic group: No


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Malate Dehydrogenase

Malate Dehydrogenase (E.C.1.1.1.37) a complex of the

apoenzyme and citrate


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Energy Balance in Krebs

Glycolysis: 4 ATP moleculesare produced , but 2 ATP'sare used in the process; so the total balance: 2 ATP's. In thisstage 2 NAD+ 's become NADH's.


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Energy Balance in Krebs

Electron Chain: Every NADH produces 3 ATP's. For 10 NADH's: 30 ATP'sare created.

Every FADH2 produces 2 ATP's.

We have 2 FADH2's: 4 ATP'sare created.


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Regulation

Many of the enzymes in the TCA cycle:

regulated by negative feedback from ATP

when the energy charge of the cell is high.


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Regulation

Also the enzymes: citrate synthase, isocitrate dehydrogenase; and alpha-ketoglutarate dehydrogenase, that regulate the first three steps of the

TCA cycle,

are inhibited by high concentrations ofATP.


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Regulation

This regulation ensures that the TCAcycle; will not oxidize excessive amount ofpyruvate;

and acetyl-CoA when ATP in the cell isplentiful.

This type of negative regulation by ATP:

by an allosteric mechanism


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The Electron Transport System


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The Electron Transport System

ofMitochondria

Embedded in the inner membrane are proteins; and complexes of molecules that are involved in

the process;

called electron transport. The electron transport system (ETS), as it is

called, accepts energy from carriers in the matrix;

and stores it to a form that can be used tophosphorylate ADP.


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LITERATURE CITED

Devlin,T.M. Textbook of Biochemistry withClinical Correlations,Fifth Edition,Wiley-LissPublications,New York, USA, 2002.

Lehninger, A. Principles of Biochemistry, Secondedition, Worth Publishers Co., New York, USA,1993.

Matthews, C.K. and van Holde, K.E.,Biochemistry, Second edition, Benjamin /Cummings Publishing Company Inc., SanFrancisco, 1996.

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