Transcript 04. Introduction to metabolism

Bioenergetics
The tiny hummingbirds can store enough fuel to fly a
distance of 500 miles without resting. This achievement is
possible because of the ability to convert fuels into the
cellular energy currency, ATP.
• Metabolism - the entire network of chemical
reactions carried out by living cells. Metabolism
also includes coordination, regulation and energy
requirement.
• Metabolites - small molecule intermediates in
the degradation and synthesis of polymers
Most organism use the same general pathway for extraction
and utilization of energy.
All living organisms are divided into two major classes:
Autotrophs – can use atmospheric carbon dioxide as a sole
source of carbon for the synthesis of macromolecules.
Autotrophs use the sun energy for biosynthetic purposes.
Heterotrophs – obtain energy by ingesting complex carboncontaining compounds.
Heterotrophs are divided into aerobs and anaerobs.
Common features of organisms
1. Organisms or cells maintain specific internal
concentrations of inorganic ions, metabolites and
enzymes
2. Organisms extract energy from external sources
to drive energy-consuming reactions
3. Organisms grow and reproduce according to
instructions encoded in the genetic material
4. Organisms respond to environmental influences
5. Cells are not static, and cell components are
continually synthesized and degraded (i.e.
undergo turnover)
A sequence of reactions that has a specific purpose
(for instance: degradation of glucose, synthesis of
fatty acids) is called metabolic pathway.
Metabolic pathway may be:
(a) Linear
(b) Cyclic
(c) Spiral pathway
(fatty acid
biosynthesis)
Metabolic pathways can be grouped into two paths –
catabolism and anabolism
Catabolic reactions - degrade molecules to create
smaller molecules and energy
Anabolic reactions - synthesize molecules for cell
maintenance, growth and reproduction
Catabolism is characterized by oxidation reactions and
by release of free energy which is transformed to ATP.
Anabolism is characterized by reduction reactions and
by utilization of energy accumulated in ATP molecules.
Catabolism and anabolism are tightly linked together
by their coordinated energy requirements: catabolic
processes release the energy from food and collect it
in the ATP; anabolic processes use the free energy
stored in ATP to perform work.
Anabolism and catabolism are coupled by energy
Metabolism Proceeds by Discrete Steps
• Multiple-step pathways
permit control of energy
input and output
Single-step vs multi-step
pathways
• Catabolic multi-step
pathways provide energy
in smaller stepwise
amounts
• Each enzyme in a multistep pathway usually
catalyzes only one single
step in the pathway
• Control points occur in
multistep pathways
A multistep enzyme
pathway releases
energy in smaller
amounts that can be
used by the cell
Metabolic Pathways Are Regulated
• Metabolism is highly regulated to permit organisms
to respond to changing conditions
• Most pathways are irreversible
• Flux - flow of material through a metabolic pathway
which depends upon:
(1) Supply of substrates
(2) Removal of products
(3) Pathway enzyme activities
Levels of Metabolism Regulation
1. Nervous system.
2. Endocrine system.
3. Interaction between organs.
4. Cell (membrane) level.
5. Molecular level
Feedback inhibition
• Product of a pathway controls the rate of its own
synthesis by inhibiting an early step (usually the first
“committed” step (unique to the pathway)
Feed-forward activation
• Metabolite early in the pathway activates an enzyme
further down the pathway
Covalent modification for enzyme regulation
• Interconvertible
enzyme activity can
be rapidly and
reversibly altered
by covalent
modification
• Protein kinases
phosphorylate
enzymes (+ ATP)
• Protein
phosphatases
remove phosphoryl
groups
Regulatory role of a protein kinase,
amplification by a signaling cascade
The initial signal may be amplified by the “cascade” nature of this signaling
Stages of metabolism
Catabolism
Stage I. Breakdown of macromolecules (proteins,
carbohydrates and lipids to respective building
blocks.
Stage II. Amino acids, fatty acids and glucose
are oxidized to common metabolite (acetyl CoA)
Stage III. Acetyl CoA is oxidized in citric acid
cycle to CO2 and water. As result reduced
cofactor, NADH2 and FADH2, are formed which
give up their electrons. Electrons are transported
via the tissue respiration chain and released
energy is coupled directly to ATP synthesis.
Glycerol
Catabolism
Catabolism is characterized by convergence of three
major routs toward a final common pathway.
Different proteins, fats and carbohydrates enter the
same pathway – tricarboxylic acid cycle.
Anabolism can also be divided into stages, however the
anabolic pathways are characterized by divergence.
Monosaccharide synthesis begin with CO2,
oxaloacetate, pyruvate or lactate.
Amino acids are synthesized from acetyl CoA, pyruvate
or keto acids of Krebs cycle.
Fatty acids are constructed from acetyl CoA.
On the next stage monosaccharides, amino acids and
fatty acids are used for the synthesis of
polysaccharides, proteins and fats.
Compartmentation of Metabolic
Processes in Cell
• Compartmentation of metabolic processes
permits:
- separate pools of metabolites within a cell
- simultaneous operation of opposing metabolic
paths
- high local concentrations of metabolites
• Example: fatty acid synthesis enzymes (cytosol),
fatty acid breakdown enzymes
(mitochondria)
Compartmentation of metabolic processes
The chemistry of metabolism
There are about 3000 reactions in human cell.
All these reactions are divided into six categories:
1. Oxidation-reduction reactions
2. Group transfer reactions
3. Hydrolysis reactions
4. Nonhydrolytic cleavage reactions
5. Isomerization and rearrangement reactions
6. Bond formation reactions using energy from ATP
1. Oxidation-reduction reactions
Oxidation-reduction reactions are those in which electrons are
transferred from one molecule or atom to another
Enzymes: oxidoreductases
-oxidases
- peroxidases
- dehydrogenases
-oxigenases
Coenzymes: NAD+, NADP+, FAD+, FMN+
Example:
2. Group transfer reactions
Transfer of a chemical functional group from one molecule to another
(intermolecular) or group transfer within a single molecule (intramolecular)
Enzymes: transferases
Examples:
Phosphorylation
Acylation
Glycosylation
3. Hydrolysis reactions
• Water is used to split the single molecule into two
molecules
Enzymes: hydrolases
- esterases
- peptidases
- glycosidases
Example:
4. Nonhydrolytic cleavage reactions
• Split or lysis of a substrate, generating a double
bond in a nonhydrolytic (without water), nonoxidative
elimination
Enzymes: lyases
Example:
5. Isomerization and rearrangement
reactions
Two kinds of chemical transformation:
1. Intramolecular hydrogen atom shifts changing the location of
a double bond.
2. Intramolecular rearrangment of functional groups.
Enzymes: isomerases
Example:
6. Bond formation reactions using energy
from ATP
• Ligation, or joining of two substrates
• Require chemical energy (e.g. ATP)
Enzymes: ligases (synthetases)
Experimental Methods
for Studying Metabolism
• Add labeled substrate to tissues, cells, and
follow emergence of intermediates. Use
sensitive isotopic tracers (3H, 14C etc)
• Verify pathway steps in vitro by using
isolated enzymes and substrates
• Study of the mutations in genes associated
with the production of defective enzymes
• Use metabolic inhibitors to identify
individual steps and sequence of enzymes in
a pathway
OXIDATIVE DECARBOXYLATION
OF PYRUVATE
Matrix of the mitochondria
contains pyruvate
dehydrogenase complex
The fate of glucose molecule in the cell
Synthesis of
glycogen
Glucose
Glucose-6phosphate
Glycogen
Pentose phosphate
pathway
Ribose, NADPH
Degradation of
glycogen
Gluconeogenesis
Glycolysis
Ethanol
Pyruvate
Acetyl Co A
Lactate
OXIDATIVE DECARBOXYLATION OF PYRUVATE
Only about 7 % of the total potential energy present in
glucose is released in glycolysis.
Glycolysis is preliminary phase, preparing glucose for
entry into aerobic metabolism.
Pyruvate formed in the aerobic conditions undergoes
conversion to acetyl CoA by pyruvate dehydrogenase
complex.
Pyruvate dehydrogenase complex is a bridge between
glycolysis and aerobic metabolism – citric acid cycle.
Pyruvate dehydrogenase complex and enzymes of
cytric acid cycle are located in the matrix of
mitochondria.
Entry of Pyruvate into the Mitochondrion
Pyruvate freely diffuses through the outer membrane of mitochondria through the channels formed by transmembrane proteins porins.
Pyruvate translocase, protein embedded into the inner
membrane, transports pyruvate from the intermembrane space
into the matrix in symport with H+ and exchange (antiport) for
OH-.
Conversion of Pyruvate to Acetyl CoA
• Pyruvate dehydrogenase complex (PDH complex) is
a multienzyme complex containing 3 enzymes, 5
coenzymes and other proteins.
Pyruvate
dehydrogenase
complex is giant,
with molecular
mass ranging
from 4 to 10
million daltons.
Electron micrograph of the
pyruvate dehydrogenase
complex from E. coli.
Enzymes:
E1 = pyruvate dehydrogenase
E2 = dihydrolipoyl acetyltransferase
E3 = dihydrolipoyl dehydrogenase
Coenzymes: TPP (thiamine pyrophosphate),
lipoamide, HS-CoA, FAD+, NAD+.
TPP is a prosthetic group of E1;
lipoamide is a prosthetic group of E2; and
FAD is a prosthetic group of E3.
The building block of
TPP is vitamin B1 (thiamin);
NAD – vitamin B5 (nicotinamide);
FAD – vitamin B2 (riboflavin),
HS-CoA – vitamin B3 (pantothenic acid),
lipoamide – lipoic acid
Pyruvate dehydrogenase complex is a classic example of
multienzyme complex
Overall reaction of pyruvate dehydrogenase complex
The oxidative decarboxylation of pyruvate catalized by
pyruvate dehydrogenase complex occurs in five steps.
Aerobic cells
use a
metabolic
wheel – the
citric acid
cycle – to
generate
energy by
acetyl CoA
oxidation
The Citric
Acid
Cycle
Synthesis of
glycogen
Glucose
Pentose phosphate
pathway
Glucose-6phosphate
Glycogen
Ribose, NADPH
Degradation of
glycogen
Gluconeogenesis
Glycolysis
Ethanol
Fatty Acids
The citric acid
cycle is the
final common
pathway for the
oxidation of fuel
molecules —
amino acids,
fatty acids, and
carbohydrates.
Pyruvate
Lactate
Acetyl Co A
Amino Acids
Most fuel
molecules
enter the
cycle as
acetyl
coenzyme A.
Names:
The Citric Acid
Cycle
Tricarboxylic
Acid Cycle
Krebs Cycle
In
eukaryotes
the reactions
of the citric
acid cycle
take place
inside
mitochondria
Hans Adolf Krebs.
Biochemist; born in Germany.
Worked in Britain. His
discovery in 1937 of the
‘Krebs cycle’ of chemical
reactions was critical to the
understanding of cell
metabolism and earned him
the 1953 Nobel Prize for
Physiology or Medicine.
An Overview of the Citric Acid Cycle
A four-carbon oxaloacetate condenses with a
two-carbon acetyl unit to yield a six-carbon
citrate.
An isomer of citrate is oxidatively
decarboxylated and five-carbon ketoglutarate is formed.
-ketoglutarate is oxidatively
decarboxylated to yield a four-carbon
succinate.
Oxaloacetate is then regenerated from
succinate.
Two carbon atoms (acetyl CoA) enter the
cycle and two carbon atoms leave the cycle
in the form of two molecules of carbon
dioxide.
Three hydride ions (six electrons) are
transferred to three molecules of NAD+, one The function of the citric acid
pair of hydrogen atoms (two electrons) is cycle is the harvesting of highenergy electrons from acetyl CoA.
transferred to one molecule of FAD.
1. Citrate Synthase
• Citrate formed from acetyl CoA and oxaloacetate
• Only cycle reaction with C-C bond formation
• Addition of C2 unit (acetyl) to the keto double bond
of C4 acid, oxaloacetate, to produce C6 compound,
citrate
citrate synthase
2. Aconitase
• Elimination of H2O from citrate to form C=C bond
of cis-aconitate
• Stereospecific addition of H2O to cis-aconitate to
form isocitrate
aconitase
aconitase
3. Isocitrate Dehydrogenase
• Oxidative decarboxylation of isocitrate to
a-ketoglutarate (a metabolically irreversible reaction)
• One of four oxidation-reduction reactions of the cycle
• Hydride ion from the C-2 of isocitrate is transferred to
NAD+ to form NADH
• Oxalosuccinate is decarboxylated to a-ketoglutarate
isocitrate dehydrogenase
isocitrate dehydrogenase
4. The -Ketoglutarate Dehydrogenase Complex
• Similar to pyruvate dehydrogenase complex
• Same coenzymes, identical mechanisms
E1 - a-ketoglutarate dehydrogenase (with TPP)
E2 – dihydrolipoyl succinyltransferase (with flexible
lipoamide prosthetic group)
E3 - dihydrolipoyl dehydrogenase (with FAD)
-ketoglutarate
dehydrogenase
5. Succinyl-CoA Synthetase
• Free energy in thioester bond of succinyl CoA is
conserved as GTP or ATP in higher animals (or ATP
in plants, some bacteria)
• Substrate level phosphorylation reaction
+
Succinyl-CoA
Synthetase
GTP + ADP
GDP + ATP
HS-
6. The Succinate Dehydrogenase Complex
• Complex of several polypeptides, an FAD prosthetic group and
iron-sulfur clusters
• Embedded in the inner mitochondrial membrane
• Electrons are transferred from succinate to FAD and then to
ubiquinone (Q) in electron transport chain
• Dehydrogenation is stereospecific; only the trans isomer is
formed
Succinate
Dehydrogenase
7. Fumarase
• Stereospecific trans addition of water to the
double bond of fumarate to form L-malate
• Only the L isomer of malate is formed
Fumarase
8. Malate Dehydrogenase
Malate is oxidized to form oxaloacetate.
Malate
Dehydrogenase
Stoichiometry of the Citric Acid Cycle
 Two carbon atoms enter the
cycle in the form of acetyl
CoA.
 Two carbon atoms leave the
cycle in the form of CO2 .
 Four pairs of hydrogen
atoms leave the cycle in four
oxidation reactions (three
molecules of NAD+ one
molecule of FAD are reduced).
 One molecule of GTP,
is formed.
 Two molecules of water are
consumed.
 9 ATP (2.5 ATP per NADH, and 1.5
ATP per FADH2) are produced during
oxidative phosphorylation.
 1 ATP is directly formed in the
citric acid cycle.
 1 acetyl CoA generates
approximately 10 molecules of ATP.
Functions of the Citric Acid Cycle
• Integration of metabolism. The citric acid cycle is
amphibolic (both catabolic and anabolic).
The cycle is involved in
the aerobic catabolism
of carbohydrates, lipids
and amino acids.
Intermediates of the
cycle are starting points
for many anabolic
reactions.
• Yields energy in the form of GTP (ATP).
• Yields reducing power in the form of NADH2 and
FADH2.
Regulation of the Citric Acid Cycle
• Pathway controlled by:
(1) Allosteric modulators
(2) Covalent modification of cycle enzymes
(3) Supply of acetyl CoA (pyruvate dehydrogenase
complex)
Three enzymes have regulatory properties
- citrate synthase (is allosterically inhibited by NADH, ATP,
succinyl CoA, citrate – feedback inhibition)
- isocitrate dehydrogenase
(allosteric effectors: (+) ADP; (-) NADH, ATP. Bacterial ICDH
can be covalently modified by kinase/phosphatase)
--ketoglutarate dehydrogenase complex (inhibition by ATP,
succinyl CoA and NADH
Regulation of the citric acid cycle
-
NADH, ATP, succinyl
CoA, citrate
Krebs Cycle is a Source of Biosynthetic Precursors
Glucose
Phosphoenolpyruvate
The citric acid cycle
provides
intermediates for
biosyntheses