A Road Map for Cellular Respiration
Cytosol
Mitochondrion
High-energy
electrons
carried
mainly by
NADH
High-energy
electrons
carried
by NADH
Glycolysis
2
Glucose
Pyruvic
acid
Krebs
Cycle
Electron
Transport
Figure 6.7
Respiration
Overview
O2 and glucose to CO2 + H2O + energy($$)
C6H12O6 + O2  6CO2 + 6H2O + 38 ATP
Glucose is highly reduced; contains energy
Oxygen receives the electrons to form energy
4 separate reactions
Glycolysis, Transition Reaction, Krebs Cycle, Electron Transport,
Requires Oxygen
Glucose
Oxygen
Carbon
dioxide
Water
Energy
Glycolysis
Most completely understood
biochemical pathway
Plays a key role in energy
metabolism by providing
significant portion of energy
utilized by most organisms
Splits the 6-C sugar (glycolysis)
Generates two molecules of
ATP per molecule of glucose
Converts two NAD+ to NADH
per molecule of glucose
Ethanol Fermentation
Lactic Acid Fermentation
Fermentation of glucose to
ethanol:
Wine making & baking both
exploit this process
From Lehninger
Principles of Biochemistry
2 Pyruvic acid
Glucose
Figure 6.8
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
Glycolysis takes place in the cytosol of cells.
Glucose enters the Glycolysis pathway by conversion to
glucose-6-phosphate.
Initially there is energy input corresponding to cleavage of
two ~P bonds of ATP.
Hexokinase
The first enzyme in the glycolysis pathway
Hexokinase undergoes a dramatic conformational change upon
binding glucose.
Two lobes of the enzyme come together to surround glucose and
exclude water from the active site.
The ATP binding site is formed after glucose binds to the enzyme.
"induced fit"
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
H
3
2
H
1
OH
Hexokinase H
OH
glucose-6-phosphate
1. Phosphorylation by Hexokinase:
Glucose + ATP  glucose-6-P + ADP
The reaction involves nucleophilic attack of the C6 hydroxyl O of
glucose on P of the terminal phosphate of ATP.
ATP binds to the enzyme as a complex with Mg++.
The reaction involves nucleophilic attack of the C6 hydroxyl O of
glucose on P of the terminal phosphate of ATP.
Mg++ interacts with negatively charged phosphate oxygen atoms,
providing charge compensation & promoting a favorable conformation of
ATP at the active site of the Hexokinase enzyme.
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
The reaction catalyzed by Hexokinase is highly spontaneous.
A phosphoanhydride bond of ATP (~P) is cleaved.
The phosphate ester formed in glucose-6-phosphate has a
lower DG of hydrolysis.
Hexokinase conformational change
PDB: 2YHX and 1HKG
The active site pocket changes shape upon binding glucose
Only then can ATP transfer its phosphoryl group to the C6
carbon, yielding Glu-6-P + ADP
glucose
Induced fit:
Glucose binding to Hexokinase
stabilizes a conformation in
which:
Hexokinase
the C6 hydroxyl of the bound glucose is close to the terminal
phosphate of ATP, promoting catalysis.
water is excluded from the active site.
This prevents the enzyme from catalyzing ATP hydrolysis, rather
than transfer of phosphate to glucose.
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
Only then can ATP transfer its phosphoryl group to
the C6 carbon, yielding Glu-6-P + ADP
An inhibitor of hexokinase
Xylose can cause a similar conformational change
But xylose does not get phosphorylated, so ATP hydrolysis is stimulated
with phorphoryl group transfer to water (water gets in!)
glucose
Hexokinase
It is a common motif for an enzyme active site to be located at
an interface between protein domains that are connected by a
flexible hinge region.
The structural flexibility allows access to the active site, while
permitting precise positioning of active site residues, and in some
cases exclusion of water, as substrate binding promotes a
particular conformation.
glucose
Hexokinase
Hexokinase in inhibited by G6P
When there are high levels of G6P, it will bind to the active
site, thus it acts like a competitive inhibitor
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 product glucose-6-phosphate:
by competition at the active site
by allosteric interaction at a separate enzyme site.
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-6phosphate] within the cell is ample.
KM
hexokinase vs. glucokinase
Both catalyze early step in breakdown of sugars
ATP
ADP + Pi
hexokinase KM: ~0.15mM glucose
glucokinase KM: ~20mM glucose
6 CH2OH
5
H
Glucokinase is
a variant of
Hexokinase
found in liver.
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
Glucokinase has a high KM for glucose.
It is active only at high [glucose].
One effect of insulin, a hormone produced when blood glucose
is high, is activation in liver of transcription of the gene that
encodes the Glucokinase enzyme.
Glucokinase is not subject to product inhibition by glucose6-phosphate. Liver will take up & phosphorylate glucose
even when liver [glucose-6-phosphate] is high.
Glucokinase,
with high KM
for glucose,
allows liver to
store glucose
as glycogen
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].
Isomerases catalyze bond rearrangement
within a molecule.
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
1CH2OH
O
5
H
H
4
OH
HO
2
3 OH
H
Phosphoglucose Isomerase
glucose-6-phosphate
fructose-6-phosphate
2. Isomerization by Phosphoglucose Isomerase:
glucose-6-P  fructose-6-P
The mechanism involves acid/base catalysis, with ring
opening, isomerization, and then ring closure.
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+
1CH2OPO32
H
H
4
OH
HO
2
3 OH
H
fructose-1,6-bisphosphate
3. Phosphorylation by Phosphofructokinase :
fructose-6-P + ATP  fructose-1,6-bisP + ADP
The Phosphofructokinase reaction is the rate-limiting
step of Glycolysis.
The enzyme is highly regulated.
Phosphofructokinase, PFK-1
Catalyzes the committing step into glycolysis
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
5
Mg2+
H
fructose-6-phosphate
1CH2OPO32
H
H
HO
3 OH
4
OH
2
H
fructose-1,6-bisphosphate
3. Phosphorylation by Phosphofructokinase :
fructose-6-P + ATP  fructose-1,6-bisP + ADP
The enzyme is highly regulated.
fructose-1,6-bisP, the product CANNOT be catabolized by any other
pathway by glycolysis
Process needs a ton of energy and is irreversible
Catalytic site
Allosteric site
Allosteric site
Catalytic site
Phosphofructokinase, PFK-1
Has five, 5, allosteric regulators
AMP, ADP, ATP, citrate and fructose 2,6-bisphosphate
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+
1CH2OPO32
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.
PFK-1 Regulation
AMP and ADP are activators. As ATP is consumed,
ADP and sometimes AMP levels build up, triggering the
need for more ATP.
The enzyme is highly regulated by ATP.
If there is a lot of ATP in the cell, then glycolysis is not
necessary.. ATP will build at an allosteric site and inhibit
binding of F6-P.
PFK Regulation
Citrate – Inhibitor of PFK-1 in liver; an early intermediate of
the citric acid cycle. Its presence indicates that the
needs of the cell are being met by other means so
glycolysis can slow down.
Fructose 2,6-bisphosphate – a powerful activator of PFK-1.
F26BP made when plenty of F6P, thus plenty of glucose
PFKs equilibrium is towards the T state so it NEEDs
F26BP to take it to R!
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.
1CH2OPO3
2C
O
HO 3C
H 4C
H
H
2
H
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
dihydroxyacetone
phosphate
5
C
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2
CH
OPO
3
2
3
glyceraldehyde-3phosphate
Triosephosphate Isomerase
4. Cleavage by Aldolase:
fructose-1,6-bisphosphate 
dihydroxyacetone-P + glyceraldehyde-3-P
The reaction is an aldol cleavage, the reverse of an aldol
condensation.
lysine
2
1CH2OPO3
H
+
H3N
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.
1CH2OPO3
2C
O
HO 3C
H 4C
H
H
2
H
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
dihydroxyacetone
phosphate
5
C
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2
CH
OPO
3
2
3
glyceraldehyde-3phosphate
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
CH2OPO32
dihydroxyacetone
phosphate
+
H
OH
H H
C
C
+
OH
CH2OPO32
enediol
intermediate
+
H
O
C
H
C
OH
CH2OPO32
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 (base) and His (acid) residues extract and
donate protons during catalysis.
http://chemistry.umeche.maine.edu/CHY431/Enzyme3.html
OH
O
HC
O
O
C
C
CH2OPO32
CH2OPO32
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.
structure is an ab barrel, or TIM barrel.
In an ab barrel there are 8 parallel b-strands surrounded by 8 ahelices with short loops connecting alternating b-strands & ahelices.
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.
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.
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
1C
H
2
C
OH
OPO32
+ 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 glyceraldehyde- 3phosphate, 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.