Transcript Karbohidrat Metabolizması

Glikoliz
.
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
Glikoliz , hücrenin sitozolunda meydana gelir.
Glukoz, glukoz-6-fosfata dönüşerek glikoliz
yoluna girer.
Başlangıçta, ATP’nin iki ~P bağının kırılmasına
bağlı olarak enerji girişi olur.
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
glucose-6-phosphate
1. Hekzokinz aşağıdaki reaksiyonu katalizler
Glukoz + ATP  glukoz-6-P + ADP
Reaksiyon, glukozun C6 hidroksil O’nin ATP’nin
terminal P’e nukleofilik atak yapmasını içerir.
ATP ,enzime Mg++ le kompleks oluşturarak bağlanır..
NH2
ATP
N
N
adenosine triphosphate
O

O
P
O
O
O
P
O
N
O
O
P
O
adenine
CH2
O
N
O
H
H
OH
H
OH
H
ribose
Mg++ negatif yüklü fosfat esteri ile etkileşir bu şekilde
ATP’nin hekzokinaz enziminin aktif merkezi için
uygun yük konformasyonunu sağlar.
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
glucose-6-phosphate
Heksokinaz la katalize edilen reaksiyon yüksek
derecede spontandır..
ATP’nin fosfoanhidrid (~P) bağı kırılır.
Glukoz-6-fosfatta oluşan fosfat ester bağı düşük DG’ya
sahiptir
glucose
Glukozun hekzokinaza
bağlanması önemli
yapısal değişime neden
olur.
Hexokinase
Bu da glukozun C6 OH’nin, ATP’nin
terminal Pi yakınlaşmasını sağlar ve aktif
bölgeden suyun çıkarılmasına neden olur..
Bu olay da ATP’nin hidrolizini önler ve P
transferine olanak sağlar.
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
OH
H
1
OH
6 CH OPO 2
2
3
1 CH2OH
O
5
H
H
4
OH
HO
2
3 OH
H
Phosphoglucose Isomerase
glucose-6-phosphate
fructose-6-phosphate
2. Fosfogluko Izomeraz reaksiyonu:
glukoz-6-P (aldoz)  fruktoz-6-P (ketoz)
Mekanizma, asit/baz katalizini içerir, halka açılması, enediolat
arametaboliti ile izomerizasyon, ve halka kapanması gözlenir.
Triozfosfat Izomeraz ile katalize edilen benzer bir reaksiyon
daha detalı olarak gösterilecektir.
Phosphofructokinase
6 CH OPO 2
2
3
O
5
H
H
4
OH
6 CH OPO 2
2
3
1CH2OH
O
ATP ADP
HO
2
3 OH
H
fructose-6-phosphate
5
Mg2+
1CH2OPO 32
H
H
4
OH
HO
2
3 OH
H
fructose-1,6-bisphosphate
3. Phosphofructokinase catalyzes:
fructose-6-P + ATP  fructose-1,6-bisP + ADP
This highly spontaneous reaction has a mechanism similar
to that of Hexokinase.
The Phosphofructokinase reaction is the rate-limiting step
of Glycolysis. The enzyme is highly regulated, as will be
discussed later.
1CH2OPO 3
2C
O
HO 3C
H 4C
H
H
5
C
2
H
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2
CH
OPO
3
2
3
dihydroxyacetone glyceraldehyde-3phosphate
phosphate
Triosephosphate Isomerase
4. Aldolase catalyzes: fructose-1,6-bisphosphate 
dihydroxyacetone-P + glyceraldehyde-3-P
The reaction is an aldol cleavage, the reverse of an aldol
condensation.
Note that C atoms are renumbered in products of Aldolase.
lysine
2
CH
OPO
2
3
1
H
+
H 3N
C
CH2
CH2
CH2
CH2

NH3
COO
2C

HO
H
H
NH (CH2)4
+
Enzyme
CH
3
C
OH
C
OH
4
5
2
CH
OPO
2
3
6
Schiff base intermediate of
Aldolase reaction
A lysine residue at the active site functions in catalysis.
The keto group of fructose-1,6-bisphosphate reacts with
the e-amino group of the active site lysine, to form a
protonated Schiff base intermediate.
Cleavage of the bond between C3 & C4 follows.
1CH2OPO 3
2C
O
HO 3C
H 4C
H
H
5
C
2
H
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2
CH
OPO
3
2
3
dihydroxyacetone glyceraldehyde-3phosphate
phosphate
Triosephosphate Isomerase
5. Triose Phosphate Isomerase (TIM) catalyzes:
dihydroxyacetone-P  glyceraldehyde-3-P
Glycolysis continues from glyceraldehyde-3-P. TIM's Keq
favors dihydroxyacetone-P. Removal of glyceraldehyde-3-P
by a subsequent spontaneous reaction allows throughput.
Triosephosphate Isomerase
H
H
C
OH
C
O
+
H H
CH2OPO 32
dihydroxyacetone
phosphate
+
H
OH
H H
C
C
+
OH
CH2OPO 32
enediol
intermediate
+
H
O
C
H
C
OH
CH2OPO 32
glyceraldehyde3-phosphate
The ketose/aldose conversion involves acid/base catalysis,
and is thought to proceed via an enediol intermediate, as
with Phosphoglucose Isomerase.
Active site Glu and His residues are thought to extract and
donate protons during catalysis.
OH
O
HC
O
O
C
C
CH2OPO 32
CH2OPO 32
proposed
enediolate
intermediate
phosphoglycolate
transition state
analog
2-Phosphoglycolate is a transition state analog that
binds tightly at the active site of Triose Phosphate
Isomerase (TIM).
This inhibitor of catalysis by TIM is similar in structure to
the proposed enediolate intermediate.
TIM is judged a "perfect enzyme." Reaction rate is limited
only by the rate that substrate collides with the enzyme.
Triosephosphate Isomerase
structure is an ab barrel, or
TIM barrel.
In an ab barrel there are
8 parallel b-strands surrounded
by 8 a-helices.
Short loops connect alternating
b-strands & a-helices.
TIM
TIM barrels serve as scaffolds
for active site residues in a
diverse array of enzymes.
Residues of the active site are
always at the same end of the
barrel, on C-terminal ends of
b-strands & loops connecting
these to a-helices.
TIM
There is debate whether the many different enzymes with
TIM barrel structures are evolutionarily related.
In spite of the structural similarities there is tremendous
diversity in catalytic functions of these enzymes and little
sequence homology.
OH
O
HC
TIM
O
O
C
C
CH2OPO 32
CH2OPO 32
proposed
enediolate
intermediate
phosphoglycolate
transition state
analog
Explore the structure of the Triosephosphate Isomerase
(TIM) homodimer, with the transition state inhibitor
2-phosphoglycolate bound to one of the TIM monomers.
Note the structure of the TIM barrel, and the loop that
forms a lid that closes over the active site after binding
of the substrate.
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
1C
H
2
C
OH
OPO 32
+ H+ O
NAD+ NADH
1C
+ Pi
H C OH
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
2
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
6. Glyceraldehyde-3-phosphate Dehydrogenase
catalyzes:
glyceraldehyde-3-P + NAD+ + Pi 
1,3-bisphosphoglycerate + NADH + H+
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
1C
H
2
C
OH
OPO 32
+ H+ O
NAD+ NADH
1C
+ Pi
H C OH
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
2
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
Exergonic oxidation of the aldehyde in glyceraldehyde3-phosphate, to a carboxylic acid, drives formation of an
acyl phosphate, a "high energy" bond (~P).
This is the only step in Glycolysis in which NAD+ is
reduced to NADH.
H
H
H3N+ C
COO
CH2
SH
cysteine
O
1C
H 2 C OH
2
3 CH2OPO3
glyceraldehyde-3phosphate
A cysteine thiol at the active site of
Glyceraldehyde-3-phosphate Dehydrogenase
has a role in catalysis.
The aldehyde of glyceraldehyde-3-phosphate
reacts with the cysteine thiol to form a
thiohemiacetal intermediate.
Enz-Cys
Oxidation to a
carboxylic acid
(in a ~ thioester)
occurs, as NAD+
is reduced to
NADH.
Enz-Cys
O
OH
HC
CH
SH
S
OH
OH
CH
CH
CH2OPO32
glyceraldehyde-3phosphate
CH2OPO32
thiohemiacetal
intermediate
NAD+
NADH
Enz-Cys
S
O
OH
C
CH
CH2OPO32
acyl-thioester
intermediate
Pi
Enz-Cys
SH
2
O3PO
O
OH
C
CH
CH2OPO32
1,3-bisphosphoglycerate
The “high energy” acyl thioester is attacked by Pi to
yield the acyl phosphate (~P) product.
H
O
H
H
C
C
NH2
+
N
O

2e + H
NH2
+
N
R
R
NAD+
NADH
Recall that NAD+ accepts 2 e plus one H+ (a hydride)
in going to its reduced form.
Phosphoglycerate Kinase
O
OPO 32 ADP ATP O
O
1C
H 2C OH
2
3 CH2OPO 3
1,3-bisphosphoglycerate
C
1
Mg2+
H 2C OH
2
3 CH2OPO 3
3-phosphoglycerate
7. Phosphoglycerate Kinase catalyzes:
1,3-bisphosphoglycerate + ADP 
3-phosphoglycerate + ATP
This phosphate transfer is reversible (low DG), since
one ~P bond is cleaved & another synthesized.
The enzyme undergoes substrate-induced conformational
change similar to that of Hexokinase.
Phosphoglycerate Mutase
O
O
C
1
O
O
C
1
H 2C OH
2
CH
OPO
2
3
3
H 2C OPO 32
3 CH2OH
3-phosphoglycerate
2-phosphoglycerate
8. Phosphoglycerate Mutase catalyzes:
3-phosphoglycerate  2-phosphoglycerate
Phosphate is shifted from the OH on C3 to the
OH on C2.
Phosphoglycerate Mutase
O
O
C
1
H 2C OH
2
CH
OPO
2
3
3
3-phosphoglycerate
histidine
O
O
H
C
1
H 2C OPO 3
3 CH2OH
2
2-phosphoglycerate
H3N+
COO
C
CH2
C
HN
CH
HC
NH
An active site histidine

side-chain participates in Pi

O
O
transfer, by donating & accepting
C
1
the phosphate.
H 2C OPO32
The process involves a
2
CH
OPO
2
3
3
2,3-bisphosphate intermediate.
2,3-bisphosphoglycerate
View an animation of the
Phosphoglycerate Mutase reaction.
Enolase
O
O
C
C
1
1
H
C
2
O
O
OPO 32
3 CH2OH
C
2
OPO 32 + H2O
3 CH2
2-phosphoglycerate phosphoenolpyruvate
9. Enolase catalyzes
2-phosphoglycerate  phosphoenolpyruvate + H2O
This Mg++-dependent dehydration reaction is inhibited
by fluoride.
Fluorophosphate forms a complex with Mg++ at the
active site.
Pyruvate Kinase
O
O
ADP ATP
C
1
C
2
O
O
C
C
1
OPO 32
3 CH2
phosphoenolpyruvate
C
2
O
O
1
OH
3 CH2
enolpyruvate
C
2
O
3 CH3
pyruvate
10. Pyruvate Kinase catalyzes:
phosphoenolpyruvate + ADP  pyruvate + ATP
This reaction is spontaneous.
PEP has a larger DG of phosphate hydrolysis than ATP.
Removal of Pi from PEP yields an unstable enol, which
spontaneously converts to the keto form of pyruvate.
glucose
Glycolysis
ATP
Hexokinase
ADP
glucose-6-phosphate
Phosphoglucose Isomerase
fructose-6-phosphate
ATP
Phosphofructokinase
ADP
fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
Glycolysis continued
glyceraldehyde-3-phosphate
NAD+ + Pi
Glyceraldehyde-3-phosphate
Dehydrogenase
NADH + H+
Glycolysis
continued.
Recall that
there are 2
GAP per
glucose.
1,3-bisphosphoglycerate
ADP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
Enolase
H2O
phosphoenolpyruvate
ADP
Pyruvate Kinase
ATP
pyruvate
Glycolysis
Balance sheet for ~P bonds of ATP:
2
 How many ATP ~P bonds expended? ________
 How many ~P bonds of ATP produced? (Remember
4
there are two 3C fragments from glucose.) ________
 Net production of ~P bonds of ATP per glucose:
________
2
Glycolysis
Balance sheet for ~P bonds of ATP:
 2 ATP expended
 4 ATP produced (2 from each of two 3C fragments
from glucose)
 Net production of 2 ~P bonds of ATP per glucose.
Glycolysis - total pathway, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
1C
H
2
C
OH
OPO 32
+ H+ O
NAD+ NADH
1C
+ Pi
H C OH
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
2
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
Fermentation
Anaerobes lack a respiratory chain for reoxidizing NADH.
They must reoxidize NADH through some other reaction.
NAD+ is needed for Glyceraldehyde-3-P Dehydrogenase
of Glycolysis.
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
O
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Skeletal muscles function anaerobically in exercise, when
aerobic metabolism cannot keep up with energy needs.
Pyruvate is converted to lactate, regenerating NAD+
needed for Glycolysis.
Glycolysis is the main source of ATP under anaerobic
conditions.
Fermentation
Pyruvate
Decarboxylase
Alcohol
Dehydrogenase
CO2
NADH + H+ NAD+
O
O
C
C
O
CH3
pyruvate
H
O
C
CH3
acetaldehyde
H
H
C
OH
CH3
ethanol
Some anaerobic organisms metabolize pyruvate to
ethanol, which is excreted as a waste product.
The Alcohol Dehydrogenase reaction regenerates
NAD+, needed for continuation of Glycolysis.
Glycolysis, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
Fermentation, from glucose to lactate:
glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP
Anaerobes excrete the product of fermentation (e.g.,
lactate or ethanol). They derive only 2 ATP from glucose
catabolism.
In aerobic organisms, pyruvate is instead oxidized
further to CO2, via Krebs Cycle and oxidative
phosphorylation, with production of additional ATP.
Glycolysis Enzyme/Reaction
DGo'
DG
kJ/mol kJ/mol
Hexokinase
Phosphoglucose Isomerase
Phosphofructokinase
Aldolase
Triosephosphate Isomerase
Glyceraldehyde-3-P Dehydrogenase
& Phosphoglycerate Kinase
-20.9 -27.2
+2.2
-1.4
-17.2 -25.9
+22.8
-5.9
+7.9 negative
-16.7
-1.1
Phosphoglycerate Mutase
Enolase
Pyruvate Kinase
+4.7
-3.2
-23.0
-0.6
-2.4
-13.9
*Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3rd Edition, John
Wiley & Sons, New York, p. 613.
Three Glycolysis enzymes catalyze spontaneous
reactions: Hexokinase, Phosphofructokinase &
Pyruvate Kinase.
Control of these enzymes determines the rate of the
Glycolysis pathway.
Local control involves dependence of enzyme-catalyzed
reactions on concentrations of pathway substrates or
intermediates within a cell.
Global control involves hormone-activated production
of second messengers that regulate cellular reactions for
the benefit of the organism as a whole.
Local control will be discussed here. Regulation by
hormone-activated cAMP signal cascade will be
discussed later.
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
glucose-6-phosphate
Hexokinase is inhibited by its product
glucose-6-phosphate.
Glucose-6-phosphate inhibits by competition at the
active site, as well as by allosteric interactions at a
separate site on the enzyme.
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
3
2
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
glucose-6-phosphate
Cells trap glucose by phosphorylating it, preventing exit
on glucose carriers.
Product inhibition of Hexokinase ensures that cells will
not continue to accumulate glucose from the blood, if
[glucose-6-phosphate] within the cell is ample.
Glucokinase, a variant of Hexokinase found in liver, has
a high KM for glucose. It is active only at high [glucose].
Glucokinase is not subject to product inhibition by
glucose-6-phosphate.
Liver will take up & phosphorylate glucose even when
liver [glucose-6-phosphate] is high.
Liver Glucokinase is subject to inhibition by glucokinase
regulatory protein (GKRP).
The ratio of Glucokinase to GKRP changes in different
metabolic states, providing a mechanism for modulating
glucose phosphorylation.
Glycogen
Glucose-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.
Glucokinase, with its high KM for glucose, allows the liver
to store glucose as glycogen, in the fed state when blood
[glucose] is high.
Glycogen
Glucose-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.
Glucose-6-phosphatase catalyzes hydrolytic release of Pi
from glucose-6-P. Thus glucose is released from the liver to
the blood as needed to maintain blood [glucose].
The enzymes Glucokinase & Glucose-6-phosphatase, both
found in liver but not in most other body cells, allow the
liver to control blood [glucose].
Phosphofructokinase
6 CH OPO 2
2
3
O
5
H
H
4
OH
6 CH OPO 2
2
3
1CH2OH
O
ATP ADP
HO
2
3 OH
H
fructose-6-phosphate
5
Mg2+
1CH2OPO 32
H
H
4
OH
HO
2
3 OH
H
fructose-1,6-bisphosphate
Phosphofructokinase is usually the rate-limiting step of
the Glycolysis pathway.
Phosphofructokinase is allosterically inhibited by ATP.
 At low concentration, the substrate ATP binds only at the
active site.
 At high concentration, ATP binds also at a low-affinity
regulatory site, promoting the tense conformation.
60
low [ATP]
PFK Activity
50
40
30
high [ATP]
20
10
0
0
0.5
1
1.5
[Fructose-6-phosphate] mM
2
The tense conformation of PFK, at high [ATP], has lower
affinity for the other substrate, fructose-6-P. Sigmoidal
dependence of reaction rate on [fructose-6-P] is seen.
AMP, present at significant levels only when there is
extensive ATP hydrolysis, antagonizes effects of high ATP.
Glycogen
Glucose-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.
Inhibition of the Glycolysis enzyme Phosphofructokinase
when [ATP] is high prevents breakdown of glucose in a
pathway whose main role is to make ATP.
It is more useful to the cell to store glucose as glycogen
when ATP is plentiful.