Bioenergetics

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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.
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• 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 carbon-containing
compounds. Heterotrophs are divided into aerobs
and anaerobs.
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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)
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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
(c) Spiral pathway (fatty acid biosynthesis)
(a) Linear (b) Cyclic
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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.
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Anabolism and catabolism are coupled by energy
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Metabolism Proceeds by Discrete Steps
Single-step vs multi-step pathways

• Multiple-step pathways permit control of energy
  input and output
• Catabolic multi-step pathways provide energy in
  smaller stepwise amounts
• Each enzyme in a multi-step 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
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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

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• Levels of Metabolism Regulation
• Nervous system.
• Endocrine system.
• Interaction between organs.
• Cell (membrane) level.
• Molecular level

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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

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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

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Regulatory role of a protein kinase,
amplification by a signaling cascade
The initial signal may be amplified by the
cascade nature of this signaling
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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.
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Glycerol
Catabolism
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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.
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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)

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Compartmentation of metabolic processes
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The chemistry of metabolism

• There are about 3000 reactions in human cell.
• 
  
  All these reactions
  are divided into six categories
• Oxidation-reduction reactions
• Group transfer reactions
• Hydrolysis reactions
• Nonhydrolytic cleavage reactions
• Isomerization and rearrangement reactions
• Bond formation reactions using energy from ATP

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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
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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
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3. Hydrolysis reactions

• Water is used to split the single molecule into
  two molecules

Enzymes hydrolases
- esterases -
peptidases
- glycosidases
Example
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4. Nonhydrolytic cleavage reactions

• Split or lysis of a substrate, generating a
  double bond in a nonhydrolytic (without water),
  nonoxidative elimination

Enzymes lyases
Example
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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
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6. Bond formation reactions using energy from ATP

• Ligation, or joining of two substrates
• Require chemical energy (e.g. ATP)

Enzymes ligases (synthetases)
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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

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OXIDATIVE DECARBOXYLATION OF PYRUVATE
Matrix of the mitochondria contains pyruvate
dehydrogenase complex
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The fate of glucose molecule in the cell
Synthesis of glycogen
Glucose
Pentose phosphate pathway
Glucose-6-phosphate
Glycogen
Ribose, NADPH
Degradation of glycogen
Gluconeogenesis
Glycolysis
Lactate
Ethanol
Pyruvate
Acetyl Co A
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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.
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Entry of Pyruvate into the Mitochondrion
Pyruvate freely diffuses through the outer
membrane of mitochon-dria 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-.
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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.
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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
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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.
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The Citric Acid Cycle
Aerobic cells use a metabolic wheel the citric
acid cycle to generate energy by acetyl CoA
oxidation
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Synthesis of glycogen
Glucose
Pentose phosphate pathway
Glucose-6-phosphate
Glycogen
Ribose, NADPH
Degradation of glycogen
Gluconeogenesis
Glycolysis
Lactate
Ethanol
Pyruvate
Fatty Acids
Amino Acids
Acetyl Co A
The citric acid cycle is the final common pathway
for the oxidation of fuel molecules amino
acids, fatty acids, and carbohydrates.
Most fuel molecules enter the cycle as acetyl
coenzyme A.
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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.
Names The Citric Acid Cycle Tricarboxylic
Acid Cycle Krebs Cycle
In eukaryotes the reactions of the citric acid
cycle take place inside mitochondria
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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 pair
of hydrogen atoms (two electrons) is transferred
to one molecule of FAD.
The function of the citric acid cycle is the
harvesting of high-energy electrons from acetyl
CoA.
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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

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2. Aconitase

• Elimination of H2O from citrate to form CC bond
  of cis-aconitate
• Stereospecific addition of H2O to cis-aconitate
  to form isocitrate

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3. Isocitrate Dehydrogenase

• Oxidative decarboxylation of isocitrate
  toa-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

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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)

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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

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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

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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

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8. Malate Dehydrogenase
Malate is oxidized to form oxaloacetate.
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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.

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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.

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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

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Regulation of the citric acid cycle
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Krebs Cycle is a Source of Biosynthetic Precursors
The citric acid cycle provides intermediates for
biosyntheses