A M A L L I A N . S E T Y A W A T I , M D , M . S I . M E D

D E P T . O F M E D I C A L B I O C H E M I S T R Y F K U N D I P

KREBS’ CYCLE

Biochemistry-lecture notes

Study objectives

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Increases awareness of Nutrition, Metabolism, and Temperature Regulation with respect to Krebs’ cycle

Understand:

-basic principles of Krebs cycle

Underlying principles in Krebs cycle with pursuant to metabolic regulation

Application of Krebs’ cycle in clinical setting & human milieu system

Establish foundation to facilitate further pursuance in related subject matter either in laboratory (research) or clinical context

TEXT & REFFERENCES

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• RECOMMENDED TEXT (either)

• 1. Murray KR., Granner DK. Harper’s illustrated Biochemistry. Lange Medical Books/McGraw-Hill.26th ed.2003:130-

• 2. Lehninger: Principles of biochemistry

USEFUL SITES:

1. http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter25/animation__how_the_krebs_cycle_works

2. http://www.wiley.com/college/pratt/0471393878/student/animations/citric_acid_cycle/index.html

SOME RECENT ARTICLES (optional)

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L. J. Reed (2001) "A trail of research from lipoic acid to a-keto acid dehydrogenase complexes," J. Biol. Chem. 276: 38329-38336. T. E. Roche, Y. Hiromasa, A. Turkan, X. Gong, T. Peng, X. Yan, S. A. Kasten, H. Bao & J. Dong (2003) "Essential roles of lipoyl domains in the activated function and control of pyruvate dehydrogenase kinases and phosphatase isoform 1," Eur. J. Biochem. 270: 1050-1056. R. A. Harris, M. M. Bowker-Kinley, B. Hyang & P. Wu (2002) "Regulation of the activity of the pyruvate dehydrogenase complex," Advan. Enzyme Regul. 42: 249-259. T. E. Roche, Y. Hiromasa, A. Turkan, X. Gong, T. Peng, X. Yan, S. A. Kasten, H. Bao & J. Dong (2003) "Essential roles of lipoyl domains in the activated function and control of pyruvate dehydrogenase kinases and phosphatase isoform 1," Eur. J. Biochem. 270: 1050-1056. H.-S. Kwon & R. A. Harris (2004) "Mechanisms responsible for regulation of pyruvate dehydrogenase kinase 4 gene expression," Advan. Enzyme Regul. 44: 109-121. V. I. Bunik (2003) "2-Oxo acid dehydrogenase complexes in redox regulation: Role of the lipoate residues and thioredoxin," Eur. J. Biochem. 270: 1036-1042. S. J. Lloyd, H. Lauble, G. S. Prasad & C. D. Stout (1999) "The mechanism of aconitase: 1.8 A resolution crystal structure of the S642A:citrate complex," Protein Science 8: 2655-2662. C. R. D. Lancaster (2002) "Succinate:quinone oxidoreductases: an overview," Biochimica et Biophysica Acta 1553: 1-6. G. Cecchini (2003) "Function and structure of complex II of the respiratory chain," Annu. Rev. Biochem. 72: 77-109. O. E. Owen, S. C. Kalhan & R. W. Hanson (2002) "The key role of anaplerosis and cataplerosis for citric acid cycle function," J. Biol. Chem. 277: 30409-30412. J. L. S. Milne, X. Wu, M. J. Borgnia, J. S. Lengyel, B. R. Brooks, D. Shi, R. N. Perham & S. Subramaniam (2006) "Molecular structure of a 9-MDa icosahedral pyruvate dehydrogenase subcomplex containing the E2 and E3 enzymes using cryoelectron microscopy," J. Biol. Chem. 281: 4364-4370. C. A. Brautigam, R. M. Wynn, J. L. Chuang, M. Machius, D. R. Tomchick & D. T. Chuang (2006) "Structural insight into interactions between dihydrolipoamide dehydrogenase (E3) and E3 binding protein of human pyruvate dehydrogenase complex," Structure 14: 611-621.

LECTURE CONDUCT & ASSESMENT

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Three hours lecture

Study assesment will be conducted:

• PRE-SEMESTER & will be highlighted in

• Clinical relevancy of Krebs’ cyle

• Medical biochemistry knowledge in Krebs’ cyle

• UKDI (competence skills assesment for medical doctor ~ Krebs’cyle)

• 80% of the exam questions will be relevant to this lecture notes & supplementary hand-out, the rest will be testing on your self-reading on the refference below

Class organization:

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Please prepare a paper sheet & write with NAME and NIM

This paper sheet will also be considered as a student presence notification

Prepare for the quizzes during the next 50 minutes’ lect

In the end of this class, the paper sheets shall be collected

In any term, the students whom do not collect the papers will be regarded as ABSENT

Outline of lectures note: KREBS CYCLE

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Introduction

Biomedical importance

Reactions of the Citric Acid Cycle

Citric acid cycle role in metabolism

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If citrate is added the rate of respiration is often increased . . . the extra oxygen uptake is by far greater than can be accounted for by the complete oxidation of citrate . . . Since citric acid reacts catalytically in the tissue it is probable that it is removed by a primary reaction but regenerated by a subsequent reaction.

—H. A. Krebs and W. A. Johnson, article in Enzymologia, 1937

Sir Hans Adolf Krebs

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A British biochemist credited with

discovering the cycle. Sir Krebs outlined the

steps of the cycle in 1937.

INTRODUCTION

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Krebs cycle citric acid cycle, tricarboxylic acid

Location mitochondria

Krebs cycle reaction:

1. oxidize acetyl residues (as acetyl-CoA)

2. reduce coenzymes that upon reoxidation are linked to the formation of ATP.

ORGANIC MOLECULES CAN BE SYNTHESIZED & BROKEN DOWN AS NEEDED

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Metabolic pool– substrates are entry points for degradation or synthesis of large molecules

CATABOLISM reactions that breakdown molecules

ANABOLISM reactions that synthesize

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The citric acid cycle serves two main purposes: To increase the cell’s ATP-producing potential by generating a reduced electron carriers such as NADH and reduced ubiquinone; and To provide the cell with a variety of metabolic precursors.

glycolysis

Citric acid

cycle =

TCA= Kreb’s

Cycle

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Cell organization

KREBS CYCLE IS AEROBIc

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This process is aerobic, requiring oxygen as the final oxidant of the reduced coenzymes.

The enzymes of the citric acid cycle are located cated in the mitochondrial matrix, either free or attached to the inner mitochondrial membrane, where the enzymes of the respiratory chain are also found.

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BIOMEDICAL IMPORTANCE

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The final common pathway aerobic oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids etabolized to acetyl-CoA or intermediates of

A central role in gluconeogenesis,lipogenesis, and interconversion of amino acids.

Mostly these reactions can tak e place in all tissue but only liver which can extend the process

Thus in any condition of large numbers of hepatic cells are damaged as in acute hepatitis or replaced by connective tissue (as in cirrhosis).

CATALYTIC ROLE OF OXALOACETATE

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The cycle starts

the acetyl-CoA + the four-carbon oxaloacetate (4

C)

Forming a six-carbon tricarboxylic acid, citrate.

In the subsequent reactions, two molecules of CO2 are released and oxaloacetate is regenerated.

A small quantity of oxaloacetate oxidation of a large quantity of acetyl-CoA; oxaloacetate may be considered to play a catalytic role.

PRINCIPLES OF KREBS CYCLE

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The citric acid cycle is an integral part of the process by which much of the free energy liberated during the Oxidation of fuels is made available.

During oxidation of acetyl-CoA, coenzymes are reduced and subsequently reoxidized in the respiratory chain, linked to the formation.

Krebs cycle is an ACTIVE & FULLY-REGULATED process in the cell.

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J A L U R R E A K S I S I K L U S K R E B ’ S

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MAIN FUNCTION :

1. Oxidized acetyl CoA CO2, H2O dan E!

(1 mol acetyl CoA releases 12 mol ATP

this cycle releases abundant H+ & electron which will enter respiratory cycle)

2. TCA cycle members are amphibolic :

can be further oxidized to become E! or

synthesized to another compounds

TCA CYCLE SERVES AS

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Are potentially energy producer:

* AA catabolism TCA cycle members E!

* fatty acid β-oxidation acetyl CoA E!

* glucosa oxidation pyruvat acetyl CoA

E!

Can be recycled to another substrates, eg :

* glucosa (gluconeogenesis)

* certain amino acid

* fatty acid (lipogenesis)

TCA CYCLE MEMBERS ARE AMPHIBOLIC

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TAHAPAN REAKSI SIKLUS ASAM SITRAT

Tahap 1. sitrat sintase

Asetil KoA + oksaloasetat + H2O sitrat + KoA-SH

Merupakan reaksi kondensasi aldol yg disertai hidroli

sis dan berjalan searah

Tahap 2

Sitrat diubah menjadi isositrat oleh enzim akonitase yg mengandung Fe++ caranya : mula2 terjadi dehidrasi menjadi cis-akonitat ( yg tetap terikat enzim )

kemudian terjadi rehidrasi menjadi isositrat

TAHAPAN REAKSI SIKLUS ASAM SITRAT

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Tahap 3

Isositrat dioksidasi menjadi oksalosuksinat (terikat en-

zim) oleh isositrat dehidrogenase yg memerlukan NAD+

Reaksi ini diikuti dekarboksilasi oleh enzim yg sama menjadi α-ketoglutarat. Enzim ini memerlukan Mn++ /

Mg++

Ada 3 jenis isozim isositrat dehidrogenase :

* satu jenis isozim menggunakan NAD+ isozim ini hanya ditemukan di dalam mitokondria NADH + H+ yg terbentuk akan diteruskan dalam rantai respi-

rasi

* Dua jenis isozim yg lain menggunakan NADP+ dan ditemukan dalam mitokondria dan sitosol

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Tahap 4

Dekarboksilasi oksidatif α-ketoglutarat (caranya seper-

ti pada dekarboksilasi oksidatif piruvat) menjadi suksi

nil KoA oleh enzim α-ketoglutarat dehidrogenase kom

pleks

Enzim ini memerlukan kofaktor seperti : TPP, Lipoat,

NAD+, FAD dan KoA-SH

Reaksi ini secara fisiologis berjalan searah

Reaksi ini dapat dihambat oleh arsenit menga

kibatkan akumulasi / penumpukan α-ketoglutarat

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Tahap 5 Suksinat thikonase

Suksinil KoA Suksinat

Reaksi ini memerlukan ADP atau GDP yg dengan Pi akan membentuk ATP atau GTP. Juga memerlukan Mg++

Reaksi ini merupakan satu2nya dalam TCA cycle yg membentuk senyawa fosfat berenergi tinggi pada ting-

kat substrat

Pada jaringan dimana glukoneogenesis terjadi ( hati &

ginjal) terdapat 2 jenis isozim suksinat thiokonase, sa-

tu jenis spesifik GDP, satu jenis untuk ADP.

Pada jaringan nonglukoneogenik hanya ada isozim yg menggunakan ADP

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Tahap 6 Suksinat dehidrogenase

Suksinat + FAD Fumarat + FADH2

Reaksi ini tdak lewat NAD, dihambat oleh malonat

Tahap 7 Fumarase

Fumarat + H2O L-Malat

Tahap 8 Malat dehidrogenase

L-Malat + NAD+ Oksaloasetat + NADH + H+

Reaksi ini membentuk kembali oksaloasetat

Reaksi total :

Asetil KoA + 3NAD+ + FAD + ADP (atau GDP) + Pi +

H2O 2CO2 + KoA-SH + 3 NADH + 3 H+ + FADH2 + ATP ( atau GTP)

Citric Acid Cycle

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Pyruvic Acid to Acetyl CoA

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Citric Acid Cycle

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Citric Acid Cycle

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Citric Acid Cycle

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Citric Acid Cycle

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1.

Synthesi

of citric acid

2.

Dehydra-

tion

3.

Hydration

Citric Acid Cycle

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4.

Oxidation

NAD+ to

electron

transport

5.

Decar-boxy-lation

Remove

CO2

Citric Acid Cycle

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6.

Oxidation

NAD+ to

electron

transport

Decar-boxy-lation

Thiol

synthesis

Citric Acid Cycle

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7.

Hydrol-ysis

Make ATP

8.

Oxidation

9.

Hydration

10.

Oxidation

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LIHAT GAMBAR SIKLUS ASAM SITRAT

REAKSI DEHIDROGENASE

* yg menggunakan NAD+ 3 ATP

* yg menggunakan FAD (tak lewat NAD+) 2 ATP

Suksinat thikonase : 1 ATP atau 1 GTP

Reaksi yg menghasilkan CO2 ( dekarboksilasi oksida-

tif) : reaksi yg dikatalisis oleh isositrat dehidrogenase dan α-ketoglutarat dehidrogenase kompleks

Vitamin B yg berperan pada TCA cycle sbg bentuk ko-

enzimnya :

Thiamin TPP

Niacin NAD

Riboflavin FAD

Asam pantotenat KoA

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JUMLAH ENERGI YANG TERBENTUK

Oksidasi 1 mol asetil KoA lewat TCA cycle menghasil-

kan :

* 3 mol (NADH + H+) yg akan masuk rantai respirasi

menghasilkan 3 x 3 mol ATP = 9 mol AP

* 1 mol FADH2 yg akan masuk rantai respirasi meng-

hasilkan 2 mol ATP

* Enzim suksinat thiokinase menghasilkan 1 mol ATP

( atau GTP )

* Jadi dari 1 mol asetil KoA dihasilkan 12 mol senyawa fosfat berenergi tinggi

PRODUK TCA CYCLE 7/26/2010 Setyawati AN (2010)

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INHIBITOR SIKLUS ASAM SITRAT

Fluoroasetat :

* Dgn KoA-SH membentuk fluoroasetil-KoA

* Fluoroasetil-KoA berkondensasi dgn oksaloasetat membentuk fluorositrat ( dikatalisis oleh sitrat sintase)

* Fluorositrat menghambat akonitase terjadi

akumulasi sitrat

* Fluoroasetat didapatkan misalnya pada pestisida

Malonat : menghambat suksinat dehidrogenase

Arsenit : menghambat α-ketoglutarat dehidrogenase

kompleks

KREBS CYCLE PARTICIPATION IN FA SYNTHESIS

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Pyruvat dehydrogenase is a mitochondrial enzyme

Acetyl CoA is the precrusor of long chains FA in nonruminants

Acetyl CoA made by pruvate dehydrogenase enzymes cannot cross mitochondria membran,

Therefore novice citrate cytosol ATP-sitrat liase enzym acetyl CoA FA in cytosol

TCA CYCLE IN FA SYNTHESIS FROM GLUCOSE :

INVOLVEMENT OF KREBS CYCLE IN GLUCONEOGENESIS

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Piruvat dehidrogenase :

* inhibited by : acetyl-CoA, NADH, ATP

* AMP : allosteric activator

TCA :

mainly controlled by NAD+ and NADH intra-mitokondrial (ratio

NAD+ dan NADH intramitokondrial)

Isositrat dehidrogenase :

* activated by ADP, inhibited by NADH

α-ketoglutarat dehidrogenase :

* inhibited by succinyl CoA

* inhibited by NADH

Malat dehydrogenase : inhibited by NADH

TCA CYCLE REGULATI ON

TCA CYCLE REGULATION

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REGULASI TCA CYCLE 7/26/2010 Setyawati AN (2010)

TCA CYCLE REGULATION

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TCA CYCLE regulation

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* Depend on demand and supply of TCA cycle.

* Fulfillment of NAD and FAD

* Restriction by NADH

* High energy sign turn off

* Low-energy signturn on

Direct reaction : citrat syntase & α-ketoglutarat dehidrogenase is one way & irreversible

TCA CYCLE REGULATION:

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CLINICAL PRESPECTIVES

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Citric Acid Cycle Summary

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Link to vchembook - ATP calculation

Citric Acid Cycle

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The Cytochrome System

6 points along pathway where hydrogen is released and temporarily bound to NAD

Reduced coenzyme NADH2 transfers hydrogen to a chain of hydrogen carriers called cytochrome system

These systems are attached to the cristate of every mitochondrion

Transfer of hydrogen from each NADH2 along system

-produces 3 ATP

-process called oxidative phosphorylation

Complete oxidation of glucose yields 38 ATP

-2 during glycolysis

-36 during oxidative phosphorylation

The role of oxygen

Final hydrogen acceptor

Combines to form water

Controlled by enzyme cytochrome oxidase

Presence of oxygen also essential for hydrogen to pass along the cytochrome system

THANK YOU !

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Shall you have any query regarding to this lecture, please do not hessitate to mail : dramallia@undip.ac.id