AMALLI A N. SETYAWATI , MD, M. SI .

MED
DEPT. OF MEDI CAL BI OCHEMI STRY FK UNDI 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. Harpers illustrated
Biochemistry. Lange Medical Books/McGraw-
Hill.26
th
ed.2003:130-
2. Lehninger: Principles of biochemistry
USEFUL SITES:
1. http://highered.mcgraw-
hill.com/sites/0072507470/student_view0/chapte
r25/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 ~
Krebscyle)
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 cells
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= Krebs
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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JALUR REAKSI SIKLUS KREB S
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MAIN FUNCTION :
1. Oxidized acetyl CoA CO
2
, H
2
O 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 -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 + H
2
O 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 -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 -ketoglutarat (caranya seper-
ti pada dekarboksilasi oksidatif piruvat) menjadi suksi
nil KoA oleh enzim -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 -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 + FADH
2
Reaksi ini tdak lewat NAD, dihambat oleh malonat
Tahap 7 Fumarase
Fumarat + H
2
O 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 +
H
2
O 2CO
2
+ KoA-SH + 3 NADH + 3 H
+
+ FADH
2
+
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.
Oxidatio
n
NAD
+
to
electron
transport
5.
Decar-
boxy-
lation
Remove
CO
2

Citric Acid Cycle
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6.
Oxidatio
n
NAD
+
to
electron
transport
Decar-
boxy-
lation
Thiol
synthesis
Citric Acid Cycle
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7.
Hydrol-
ysis
Make
ATP
8.
Oxidation
9.
Hydratio
n
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 CO
2
( dekarboksilasi oksida-
tif) : reaksi yg dikatalisis oleh isositrat dehidrogenase
dan -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 FADH
2
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
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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 -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
-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
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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 & -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 NADH
2
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 NADH
2
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