Metabolism

catabolism –
degradation of nutrients to generate energy and
starting materials

anabolism –
biosynthesis of biomolecules from starting
materials

metabolites –
substrates,intermediates and products of
metabolism
 Stages of Catabolism
STAGE

I­ Complex 
to Building 
Blocks

II­ Building 
Catabolic pathways Blocks to 
tend to converge. Acetyl CoA

Each arrow represents a III­ 

Oxidation 
catabolic pathway. of Acetyl 
CoA
 POLYSACCHARIDES
LIPIDS PROTEINS
glucose
fatty
acids Glycolysis amino acids
ATP
pyruvate

acetyl-CoA
FADH2 and NADH
O2 (reducing power)

oxidative tricarboxylic
phosphorylation acid (TCA)
cycle

ATP
 Coenzyme A

• performs a vital role by transporting acetyl

groups from one substrate to another
• the key to this action is the reactive
thioester bond in the acetyl form of CoA
• the thioester bond is stable enough that it
can survive inside the cell, but unstable
enough that acetyl-CoA can readily
transfer the acetyl group to another
molecule
 O
H2
O N H2C C S C
H
C H3

Phosphorylated Pantothenic Mercaptoethylamine Acyl

ADP Acid Group

CoEnzyme A
 Glycolysis

Glycolysis takes place in the cytosol of cells.

Glucose enters the Glycolysis pathway by
conversion to glucose-6-phosphate.

Glucose

10 steps 3 regulated steps

No O2
Pyruvate Lactate
 Glycolysis

Glucose + 2 ADP + 2 phosphate + 2 NAD+

2 Pyruvate + 2 ATP + 2 NADH + H2O

• Metabolic pathways are irrereversible
• Every pathway has a first committed step
• All metabolic pathways are regulated
• Metabolic pathways in eukaryotic cells
occur in specific cellular locations (gene
clusters)
Reaction of pyruvate dehydrogenase complex (PDC)

Irreversible

acetyl-CoA cannot be converted back to pyruvate;

hence “fat cannot be converted to carbohydrate”

 Pathway Enzymes

Kinase: transfers a phosphate group from ATP (i.e.

hexokinase, galactose kinase, pyruvate kinase)

Isomerase: converts one isomer to another (i.e.

phosphoglucoisomerase, triose phosphate isomerase)

Aldolase: catalyzes aldol condensation(i.e. aldolase,

functions in reverse in glycolysis)

Dehydrogenase: removes hydrogens by oxidation. Usually

require NAD+ or FAD as co-factors/co-substrates)
 Pathway Enzymes

Mutase: group transfer enzyme. Common use is to move

phosphates to different positions on sugars (i.e.
phosphoglycerate mutase, glucose-1-P mutase).
Enolase: converts C=C group to alcohol. No change in
oxidation state.
Synthase: (also known as synthetase). Usually an
enzyme that combines two things to make a new
compound. (i.e. citrate synthase, succinyl CoA
synthetase).
ATPase: Hydrolyses ATP to ADP and Pi. This reaction
runs in reverse in FoF1 ATPase to generate ATP using
the free energy of the proton gradient.
 6 CH2OH 6 CH OPO 2−
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose glucose-6-phosphate

Hexokinase catalyzes:
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++.
 NH2

ATP N
N
adenosine triphosphate
N
O O O N
−
O P O P O P O CH2 adenine
O
− − − H H
O O O
H H
OH OH
ribose

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 6 CH OPO 2−
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose 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 ∆G of hydrolysis.
 glucose

Hexokinase
Induced fit:
Binding of glucose to Hexokinase promotes a large
conformational change by stabilizing an alternative
conformation in which:
 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.
Phosphofructokinase catalyzes:
fructose-6-P + ATP  fructose-1,6-bisP + ADP
This highly spontaneous reaction.
The Phosphofructokinase reaction is the rate-limiting
step of Glycolysis.
The enzyme is highly regulated.
 Pyruvate Kinase

O O− O O− O O−
C ADP ATP C C
1 1 1

2
C OPO32− C
2
OH 2
C O

3 CH2 3 CH2 3 CH3

phosphoenolpyruvate enolpyruvate pyruvate

Pyruvate Kinase catalyzes:

phosphoenolpyruvate + ADP  pyruvate + ATP
 Glyceraldehyde-3-phosphate
Dehydrogenase
H O + H+ O OPO32−
1C NAD+ NADH 1C
+ Pi
H C OH H C OH
2 2
2− 2−
3 CH2 OPO 3 3 CH2 OPO 3

glyceraldehyde- 1,3-bisphospho-
3-phosphate glycerate

Glyceraldehyde-3-phosphate Dehydrogenase
catalyzes:
glyceraldehyde-3-P + NAD+ + Pi 
1,3-bisphosphoglycerate + NADH + H+
 H O
H
1C
H3N+ C COO−
H 2 C OH
CH2 2−
3 CH2OPO3
SH glyceraldehyde-3-
cysteine phosphate

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 SH O OH

HC CH CH2OPO32−
glyceraldehyde-3-
OH OH phosphate

Oxidation to a Enz-Cys S CH CH CH2OPO32−

NAD+ thiohemiacetal
carboxylic acid intermediate
NADH
(in a ~ thioester)
O OH
occurs, as NAD+
Enz-Cys S C CH CH2OPO32−
is reduced to acyl-thioester
Pi
NADH. intermediate
O OH
2−
Enz-Cys SH O3PO C CH CH2OPO32−
1,3-bisphosphoglycerate

The “high energy” acyl thioester is attacked by Pi to

yield the acyl phosphate (~P) product.
 H O O
H H
C C
NH2 NH2

+
N − + N
2e + H
R R
NAD+ NADH

Recall that NAD+ accepts 2 e− plus one H+ (a hydride)

in going to its reduced form.
 Lactate Dehydrogenase
O O− O O−
C NADH + H+ NAD+ C
C O HC OH
CH3 CH3
pyruvate lactate

Lactate is also a significant energy source for neurons

in the brain.
Astrocytes, which surround and protect neurons in the
brain, ferment glucose to lactate and release it.
Lactate taken up by adjacent neurons is converted to
pyruvate that is oxidized via Krebs Cycle.
 6 CH OPO 2−
2 3
5 6 CH OPO 2− 1 CH2OH
H O H 2 3
O
H
4 H 1 5 H HO 2
OH
OH OH H 4 3 OH
3 2
OH H
H OH
Phosphoglucose Isomerase
glucose-6-phosphate fructose-6-phosphate

Phosphoglucose Isomerase catalyzes:

glucose-6-P (aldose)  fructose-6-P (ketose)
The mechanism involves acid/base catalysis, with ring
opening, isomerization via an enediolate intermediate,
and then ring closure.
 Enolase
−
O H+ − −
O OH− O−
O O O
C C 1
C
1
H 2 C OPO32− C OPO32− 2C OPO32−

3 CH2OH CH2OH 3 CH2

2-phosphoglycerate enolate intermediate phosphoenolpyruvate

Enolase catalyzes:
2-phosphoglycerate  phosphoenolpyruvate + H2O
This dehydration reaction is Mg++-dependent.
2 Mg++ ions interact with oxygen atoms of the substrate
carboxyl group at the active site.
The Mg++ ions help to stabilize the enolate anion
intermediate that forms when a Lys extracts H+ from C #2.
 Phosphoglycerate Mutase
O O− O O−
C
1
C
1
H 2C OH H 2C OPO32−
2−
3 CH2 OPO 3 3 CH2OH
3-phosphoglycerate 2-phosphoglycerate

Phosphoglycerate Mutase catalyzes:

3-phosphoglycerate  2-phosphoglycerate

Phosphate is shifted from the OH on C3 to the

OH on C2.
 Phosphoglycerate Mutase
O− O−
histidine
O O
H
1
C 1
C
2− H3N+ C COO−
H 2C OH H 2C OPO3
2− CH2
3 CH2OPO3 3 CH2OH
3-phosphoglycerate 2-phosphoglycerate C
HN CH

HC NH
An active site histidine side-chain +

participates in Pi transfer, by
O O−
donating & accepting phosphate. C
1
The process involves a H 2C OPO32−
2,3-bisphosphate intermediate. 2−
3 CH2OPO3

View an animation of the 2,3-bisphosphoglycerate

Phosphoglycerate Mutase reaction.
 2−
1CH2OPO3

2C O
H O
2−
HO 3C H Aldolase 3
CH2 OPO 3 1C

H 4C OH 2C O + H 2C OH
2−
H C OH 1CH2OH 3 CH2OPO3
5
2− dihydroxyacetone glyceraldehyde-3-
6 CH2 OPO 3
phosphate phosphate
fructose-1,6-
bisphosphate
Triosephosphate Isomerase
Aldolase catalyzes:
fructose-1,6-bisphosphate 
dihydroxyacetone-P + glyceraldehyde-3-P
The reaction is an aldol cleavage, the reverse of an aldol
condensation. C atoms are renumbered in products of
Aldolase.
 lysine 1 CH 2OPO3
2−
H
− 2C NH (CH2)4 Enzyme
H3N+
C COO +
HO 3
CH
CH2
H 4
C OH
CH2
H 5
C OH
CH2 2−
6 CH 2OPO3
CH2
Schiff base intermediate of
+ NH3 Aldolase reaction

A lysine residue at the active site functions in catalysis.

The keto group of fructose-1,6-bisphosphate reacts with
the ε-amino group of the active site lysine, to form a
protonated Schiff base intermediate.
Cleavage of the bond between C3 & C4 follows.
 2−
1CH2OPO3

2C O
H O
2−
HO 3C H Aldolase 3
CH2 OPO3 1C

H 4C OH 2C O + H 2C OH
2−
H C OH 1CH2OH 3 CH2 OPO 3
5
2− dihydroxyacetone glyceraldehyde-3-
6 CH2 OPO 3
phosphate phosphate
fructose-1,6-
bisphosphate
Triosephosphate Isomerase
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 OH H
+
O
+ + +
H C OH H H C H H C
C O C OH H C OH
CH2OPO32− CH2OPO32− CH2OPO32−

dihydroxyacetone enediol glyceraldehyde-

phosphate intermediate 3-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.
 Glycolysis glucose
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
 Experimental approaches to study
metabolism

• Sequence of reaction pathway

• Mechanistic analysis, metabolic pathway
inhibition
• Regulation of pathway mechanism
 6 CH2OH 6 CH OPO 2−
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose 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 6 CH OPO 2−
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose 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
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P 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 when blood [glucose] is
high.
 Glycogen Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P 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 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.
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
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P 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.