Download Bioenergetics and High Energy Compounds

Bioenergetics and High Energy
Compounds
Dr. Sugandhika Suresh
Department of Biochemistry
• Bioenergetics is the study of how organisms
manage their energy resources
• Energy is the capacity to cause change
• It exists in various forms, some of which can
perform work
– Chemical energy is potential energy available
for release in a chemical reaction
• Energy can be converted from one form to
another
Summary of Animal Metabolism
Hydrolyse
Monosaccharides, Amino acids,
Fatty acids
Food
CHO, Protein, Fat
Anabolism
Biosynthesis
Growth,
Substances
Cell
ATP, NADH
Energy
ATP
CO2
Movement
(mechanical) Muscle
Active
Transport
(Osmotic)
O
XI
DI
S
E
Bioluminescence
(Light Emission)
Electrical energy Nerve
Transmission
• The free-energy change of a reaction tells us
whether the reaction occurs spontaneously
• A living system‟s free energy is energy that can do
work when temperature and pressure are uniform,
as in a living cell
Free-Energy Change, G
• The change in free energy (∆G) during a process is
related to the change in enthalpy, or change in total
energy (∆H), and change in entropy (T∆S):
• Enthalpy will tell us the heat content (energy)
of a system
• Entropy will tell us whether a process is
favourable (spontaneous)
For any process (A
B) at constant
pressure and temperature
The free energy change is defined as:
∆G = ∆H – T∆S
(T = absolute temperature)
Free Energy and Metabolism
• The concept of free energy can be applied to the
chemistry of life‟s processes
• Reactions in a closed system eventually reach
equilibrium and then do no work
• Cells are not in equilibrium; they are open systems
experiencing a constant flow of materials
• A catabolic pathway in a cell releases free energy
in a series of reactions
A reaction can occur spontaneously (is
favoured) only if ∆G is negative
If: ∆G is negative (-)
• the process is exergonic
• the reaction proceeds with the release of
free energy
• the reaction will be thermodynamically
favourable in the direction written
LE 8-6a
Free energy
Reactants
Amount of
energy
released
(G < 0)
Energy
Products
Progress of the reaction
Exergonic reaction: energy released
A reaction cannot occur spontaneously (is
not favoured) if ∆G is positive
If: ∆G is positive (+)
• the process is endergonic
• an input of free energy is required to
drive the reaction
• the reaction will be thermodynamically
unfavoured
(reverse process is favoured)
LE 8-6b
Free energy
Products
Energy
Reactants
Progress of the reaction
Endergonic reaction: energy required
Amount of
energy
required
(G > 0)
A system is at equilibrium and no net change
can take place if ∆G is zero
If: ∆G is zero (0)
•The process is at equilibrium
•No net flow in either the forward or the
reverse direction
•Neither process is favoured
Most of these metabolic reactions are not
spontaneous
•
•(i.e., they are accompanied by a positive
change in free energy, ΔG>0) and do not
occur without some other source of free
energy.
•
Hence, the body needs some sort of
"free-energy currency," a molecule that can
store and release free energy when it is
needed to power a given biochemical
reaction.
Just as purchasing transactions do not occur without monetary currency,
reactions in the body do not occur without energy currency
High Energy Compounds
• Compounds with a high energy bond
• If the bond is hydrolysed – chemical
energy is made available
• Less favourable reactions can be driven
forward
• ATP – most commonly encountered
The free energy change for the hydrolysis of ATP is large and negative
•This equilibrium lies so far to the right
that ATP hydrolysis can be considered
essentially irreversible
•compounds that can undergo reactions
with a resulting large negative free energy
change (like ATP) are used as shuttles of
free energy in the cell
•(the bonds are said to contain potential
transfer energy)
• Because the free-energy changes of sequential
reactions are additive;
• Any phosphorylated compound can be synthesized
by coupling the synthesis to the breakdown of
another phosphorylated compound with a more
negative free energy of hydrolysis
(1)
(2)
Hydrolysis of PEP:
Phosphorylation of ADP:
PEP
ADP + Pi
Coupled phosphorylation of ADP by PEP: PEP + ADP
pyruvate + Pi
ATP
∆G o’ = - 62 kJ/mol
∆G o’ = + 31 kJ/mol
pyruvate + ATP
∆G o’ = -31 kJ/mol
cleavage of Pi from phosphoenolpyruvate (PEP)
releases more free energy
Coupling of biochemical reactions
These two reactions share an
intermediate (Pi) and may be
expressed as sequential
(coupled) reactions
Glucose + Pi
ATP + H20
ATP + glucose
glucose 6- phosphate
ADP + Pi
∆Go’ = + 13.8 kJ/mol
∆Go’ = - 30.5 kJ/mol
ADP + glucose 6-phosphate
∆Go’ = -16.7 kJ/mol
The energy stored in the bonds of ATP is used to drive the synthesis of glucose 6phosphate even though its formation from glucose and phosphate is endergonic
In thermodynamic calculations, all that matters is the state of the system at the
beginning of the process and its state at the end
Note the range of
potential transfer
energy of these
compounds (-60 to –10
∆Go’ kJ/mol )
This indicates that some
of these phosphate
hydrolysis reactions are
very high energy
processes while others
are not.
Q: WHY do some
compounds have a
higher free energy of
hydrolysis?
It’s not the phosphate
bonds themselves, but
rather some property of
both the reactants and
products in the reactions
that contribute to the
differences in free
energy
The free energy of phosphate
hydrolysis of ATP when compared to
other molecules is at the middle.
This allows ATP to accept energy from
high energy donor compounds & to act
as a donor for low energy phosphate
acceptor.
Functions carried out by ATP
• Active transport - molecules & ions
• Mechanical work – muscle contraction &
other cellular movement
• Biosynthesis of macromolecules
(ATP serve as the immediate donor of free
energy in biological systems)
Compounds with equal free energy of
hydrolysis
1. Nucleoside triphosphates/ Deoxy
nucleoside triphosphates:
GTP ATP CTP UTP
(PPi bonds present in all compounds)
Nucleoside diphosphate forms:
ADP GDP CDP UDP
(ATP + GDP
GTP + ADP)
Compounds with more Energy than ATP
SUPER HIGH ENERGY COMPOUNDS
2. Creatine phosphate (CP)
store of free energy in muscle
CP + ADP
ATP + creatine
creatine kinase
3. PEP (glycolysis)
PEP + ADP
ATP + pyruvate
ATP – Uses in Medical Context
Active Transport
•Absorption
•HCl
•I-
of glucose in intestine
secretion in stomach
Pump in thyroid
Muscle Contraction
Energy in ATP
Helix
Random coil of Myosin Head
In all cases
ATP
Energy = 7 -14 kcal.mole-1
ATPase
ADP + Pi
27
4.
NuDP Sugars (activated molecules)
• UDP glucose
• CDP glucose
• ADP glucose
• UDP galactose
5.
Glucose derivative polymers
Biosynthesis
NuDP Bases
• CDP choline
Biosynthesis of
phospholipids
All involved in Biosynthesis = Anabolism
6.
Other Coenzyme A Esters
•Succinyl Co A
•Malonyl CoA
Cofactors
Dr. Sugandhika Suresh
Dept. of Biochemistry
30
• Small non-protein helpers of enzymes
• Enzyme is not biologically active without
the cofactor
• Without the cofactor, the enzyme is known
as the apo enzyme
31
• Apo enzyme = enzyme – (minus) prosthetic
group
Holo enzyme = enzyme + all cofactors
= core enzyme + coenzyme
eg: RNA polymerase + σ factor
cofactor
32
Eg. carboxypeptidase
33
Cofactors (small non – protein units)
Loosely bound
Inorganic
Mn2+
Mg2+
Organic
Cosubstrate
Coenzyme
Tightly bound
Prosthetic groups
Organic
Inorganic
34
Coenzymes
• Heat stable, low molecular weight organic compounds
• Gets transiently associated with the enzyme during
enzyme activity
• Mostly linked to the enzyme by non-covalent forces.
Prosthetic group
• Form tight covalent bonds, with the enzymes
–.
35
Coenzymes
• In oxidation-reduction reactions, coenzymes
often remove electrons from the substrate
and pass them to different enzymes.
• In this way, coenzymes serve to carry energy
in the form of electrons (or hydrogen atoms)
from one compound to another.
• Many coenzymes are derived from the
vitamin B complex.
36
37
• Co-substrates:
– ATP
– NAD+
– NADP+
– FMN
– FAD
Changes at the end of a reaction
38
Co-substrates
An example of a Co-substrate reaction
(i) Glucose + ATP
G6P + ADP
gives energy
(ii) Lactate + NAD+
NADH + H+ + PYRUVATE
gives reducing power
In both cases,
co-substrate is not the same, at the end of the
reaction.
39
Prosthetic groups
Inorganic prosthetic
groups
Organic prosthetic
groups
Ion
Enzyme
Co2+
Carboxypeptidase
Zn2+
Many enzymes
Se
Glutathione reductase
Cu2+
Caeruloplasmin
Haem
Hb, Mb, Cytochromes
(Porphyrin)
CHO
Glycoproteins
Lipids
Lipoproteins
40
Metabolism –
All the Biochemical Pathways in Cells
Metabolism
Anabolism
Catabolism
Anabolism
Catabolism
1. Energy using
1. Energy yielding
2. Reductive
2. Oxidative
3. Biosynthetic
3. Degradative
41
Metabolism:
the highly integrated network of
chemical reactions by which living cells
grow & sustain themselves.
The network is composed of two major
types of pathways:
1) anabolic p.w (anabolism)
2) catabolic p.w (catabolism)
42
Anabolism:
Process by which large molecules are
synthesized using smaller molecules
and energy stored in adenosine
triphosphate (ATP)
Catabolism:
Process by which the nutrients & cell
constituents are degraded to produce
energy (as ATP) & also raw materials
for anabolic reactions
43
Catabolism and anabolism
44
2 metabolic networks have 3 major functions:
(1) extract energy from nutrients
(2) synthesize building blocks that make up
large molecules of life: proteins, fats,
carbohydrates, nucleic acids &
combinations of these substances
(3) synthesize & degrade molecules required
for special functions in the cell
45
 Anabolic & catabolic reactions are
enzymatic reactions organized into
multi-step pathways (metabolic
pathways).
• Reaction sequences are composed of
many enzymatic reactions, each one
creating a product that becomes the
substrate for the subsequent enzyme.
(Enz – lower activation energy)
46
Metabolic pathway
E - enzyme
E1
E2
A
B
C
E3
E4
D
E5
E
F
metabolic intermediates or metabolites
• Most reactions of anabolic or catabolic
pathways are reversible with few
unidirectional or irreversible reactions
which control the pathway.
47
1. Metabolic pathways are irreversible.
irreversible reaction (s) of a pathway
makes the path way irreversible
2. Catabolic & anabolic pathways differ
Catabolic p.w
C
A
B
Anabolic p.w
X
Y
Why ?
48
A
B
exergonic (free energy released)
- ΔG
B
A
endergonic (free energy required)
+ ΔG
The two pathways differ at least in one
reaction
Independent control (regulation ) of
the two pathways
49
3. Every metabolic pathway has a
committed step
• an irreversible reaction early in the
pathway (exergonic) (ΔG – ve)
commits
the intermediate to continue down the
pathway
• Most reactions of a pathway function
close to equilibrium
50
4. All metabolic pathways are regulated
by laws of supply & demand
Most are controlled by regulating the
enzymes that catalyze rate limiting steps
“POINTS OF CONTROL”
First committed step often is one of the rate
limiting steps.
Prevent unnecessary synthesis of
metabolites further down the pathway
51
Eg: a) allosteric control
feed back inhibition
E1
E2
E3
A
B
C
D
E4
E
E5
F
b) covalent modification
glycogen phosphorylase
5. Metabolic pathways occur in specific
cellular locations (compartmentation)
52
Catabolism:
different proteins, polysaccharides &
lipids are broken down into relatively
few catabolic end products.
Convergent
53
Stages in the
extraction of
energy from
foodstuff
i) Large molecules
smaller units.
ii) small molecules into a few
simple units that play a central
role in metabolism. Some ATP
generated.
iii) TCA cycle &
oxidative
phosphorylation –
FINAL COMMON p.w
in oxidation of fuel
molecules.
> 90% of ATP from
54
food released
int
Anabolism:
relatively few biosynthetic precursor
molecules are used to synthesize a
large number of different proteins,
polysaccharides & lipids.
divergent
55
Summary
56
Electron Transfer Chain
Dr. Sugandhika Suresh
Dept. of Biochemistry
• An overview of cellular respiration
High-energy electrons
carried by NADH
GLYCOLYSIS
Glucose
Cytoplasmic
fluid
Figure 6.8
Pyruvic
acid
KREBS
CYCLE
ELECTRON
TRANSPORT CHAIN
AND CHEMIOSMOSIS
Mitochondrion
During cellular metabolism;
Carbohydrates
Fat
CO2 + H2O
Amino acids
NADH / FADH2
• Energy stored as NADH / FADH2.
• These co-enzymes are further oxidized to free the
energy.
• If oxidized by a single step
„E‟ lost.
• A specialized set of electron carriers are used to
free the energy and store them in ATP.
(electron transfer chain)
The Electron Transport Chain
•
A “specialized set of electron carriers”
•
Location: inner mitochondrial membrane.
•
Electrons in the NADH and FADH2
donate electrons to these carriers which
pass them down to one another.
glycolysis
inner
membrane
outer
membrane
electron
transport
chain
Krebs
cycle
H+
e-
O2
outer
compartment
H2O
Location
inner compartment
Of ETC
Transport across mitochondrial membrane
• NADH produced in glycolysis must be
transported to the mitochondrial matrix.
MITOCHONDRIAL MEMBRANE TRANSPORT
•Adenine nucleotide translocase
–Charge across the membrane determines specificity
•Phosphate carrier
–Proton symport
•Di and tri-carboxylic acid carriers
–Proton symporters
TRANSPORT OF REDUCING
EQUIVALENTS OF NADH
•Glycerol phosphate shuttle
–Two moles of ATP per mole of cytosolic NADH
•Aspartate/malate shuttle
–Three moles of ATP per mole of NADH
•
To be an electron carrier the molecules
must be able to exist in 2 forms.
1. Oxidized (before accepting electrons)
2. Reduced (after accepting electrons)
A 2+
Oxidation
A 3+
B 2+
Reduction
B+
Redox Pair
Oxidation-Reduction potentials of the complexes
• In the ETC the electron carriers are
organized into groups (multienzyme
complexes).
• ETC has 5 such complexes.
– Complex I, - NADH dehydrogenase
– Complex II, - Succinate dehydrogenase
– Complex III – Cytochrome reductase
– Complex IV- Cytochrome oxidase
– Complex V – ATP synthase
Each complex has a series of electron carriers
Sequence of electron carriers in the respiratory chain
Complex I
proton pump
Coenzyme Q
electron shuttle
Complex II, does not
pump protons
Complex III
proton pump
Cytochrome c
electron shuttle
Complex IV
proton pump
Complex I(NADH Dehydrogenase)
Takes H from NADH (if you remember
from the TCA cycle, NAD+ takes one
hydrogen ion and one hydride ion).
The H+ ions go to a molecule of FMN
which is closely associated to Complex
I, turning it into FMNH2
Coenzyme Q then diffuses over to
take the hydrogens from FMNH2, and
the complex returns to normal, ready
to accept hydrogens from the next
NADH.
CoQ is also reduced by the electrons obtained from the NADH, enabling it
to transfer electrons somewhere else
Complex II
Instead of FMN , FAD which is tightly bound to
the complex, and protein complex does not
span the entire membrane
in the TCA cycle and the Beta-Oxidation Cycle,
FADH2 can be produced from reactions involving a
couple of different enzymes (e.g. succinate
dehydrogenase, malate dehydrogenase or
acyl CoA dehydrogenase).
substrate such as malate may come along and
donate its hydrogens to FAD, forming FADH2,
which then passes its H+ ions on to coenzyme Q.
Electrons are also transfered to CoQ, reducing it
Cytochromes Complexes
III(b) ,C ,IV(a)
cytochromes contain a
ferric ion (Fe3+) which can
be reduced to a ferrous ion
(Fe2+). This is achieved by
the transfer of electrons, as
the electrons picked up by
CoQ from the other
complexes are passed on.
Cytochrome b
Complex III
Cytochrome C
Complex IV,
Cytochrome a + a3
Since cytochrome a + a3 has copper
atoms bound to it, it is able to facilitate
the production of water
What is oxidative phosphorylation?
• involves phosphorylating ADP (to produce
ATP) using oxidation reactions.
• Building up a proton gradient across the inner
membrane of mitochondria,
Chemisosmosis and oxidative phosphorylation
• Energy is not synthesized directly.
• The „E‟ generated at the complexes I, III and IV are
used to pump H+ across the inner mitochondrial
membrane, into the inter membranous space.
inter-membrane
space
The [H+] in the space increases.
This creates an electrochemical gradient
(protein motive force) across the inner
mitochondrial membrane.
cytosol
• H+ re-enters the mitochondrial matrix through the
ATP synthase molecule.
• This re-entry provides the energy for the ATP
synthase to synthesize ATP.
• Therefore ATP synthesis and e-transport are
coupled.
Brown Adipose Tissue (BAT)
• The energy generated in ETC is used for
thermogenesis (heat generation) rather
than ATP generation.
• Newborns (high BAT – produces heat)
• Hibernating animals
• In animals exposed to cold.
THERMOGENESIS
•Uncoupled mitochondria
–Brown fat for heat production
•Thermogenin
• Uncoupling protein
Inhibitors of electron transport
•
Rotenone (a plant toxin used by Amazonian
Indians to poison fish and also an insecticide),
amytal (a barbiturate),
piericidin A (a structural analog of ubiquinone)
inhibit at complex I.
•
Antimycin A ( a Streptomyces antibiotic)
inhibits complex III.
•
CN-, CO, and N3- inhibit complex IV.
Certain poisons interrupt critical
events in cellular respiration
Rotenone
Cyanide,
carbon monoxide
ELECTRON TRANSPORT CHAIN
Figure 6.13
Oligomycin
ATP SYNTHASE
HOW UNCOUPLERS WORK
•Uncouplers are weak acids
–Become protonated
–Carry protons across the membrane
–Dissipate the proton gradient
•Ionophores such as valinomycin
–Carry ions (K+) across the membrane
–Dissipate the membrane potential
Summary
Enzymes as Biological Catalysts
• Enzymes are
proteins that
increase the rate of
reaction by
lowering the energy
of activation
• They catalyze
nearly all the
chemical reactions
taking place in the
cells of the body
• Enzymes have
unique threedimensional
shapes that fit the
shapes of reactants
(substrates)
Naming Enzymes
• The name of an enzyme identifies the reacting
substance
- usually ends in –ase
• For example, sucrase catalyzes the hydrolysis of
sucrose
• The name also describes the function of the enzyme
• For example, oxidases catalyze oxidation reactions
• Sometimes common names are used, particularly for
the digestion enzymes such as pepsin and trypsin
• Some names describe both the substrate and the
function
• For example, alcohol dehydrogenase oxides ethanol
Classification of Enzymes
• Enzymes are classified according to the type of
reaction they catalyze:




Class
Oxidoreductases
Transferases
Hydrolases
Lyases
 Isomerases
 Ligases
Reactions catalyzed
Oxidation-reduction
Transfer groups of atoms
Hydrolysis
Add atoms/remove atoms
to/from a double bond
Rearrange atoms
Use ATP to combine
molecules
Oxidoreductases, Transferases and Hydrolases
Lyases, Isomerases and Ligases
Active Site of an Enzyme
• The active site is a
region within an enzyme
that fits the shape of
substrate molecules
• Amino acid side-chains
align to bind the
substrate through Hbonding, salt-bridges,
hydrophobic interactions,
etc.
• Products are released
when the reaction is
complete (they no longer
fit well in the active site)
Enzyme Specificity
• Enzymes have varying degrees of specificity for
substrates
• Enzymes may recognize and catalyze:
- a single substrate
- a group of similar substrates
- a particular type of bond
Lock-and-Key Model
• In the lock-and-key model of enzyme action:
- the active site has a rigid shape
- only substrates with the matching shape can fit
- the substrate is a key that fits the lock of the active
site
• This is an older model, however, and does not work
for all enzymes
Induced Fit Model
• In the induced-fit model of enzyme action:
- the active site is flexible, not rigid
- the shapes of the enzyme, active site, and substrate
adjust to maximumize the fit, which improves catalysis
- there is a greater range of substrate specificity
• This model is more consistent with a wider range of
enzymes
Enzyme Catalyzed Reactions
• When a substrate (S) fits properly in an active site, an
enzyme-substrate (ES) complex is formed:
E + S  ES
• Within the active site of the ES complex, the reaction
occurs to convert substrate to product (P):
ES  E + P
• The products are then released, allowing another
substrate molecule to bind the enzyme
- this cycle can be repeated millions (or even more)
times per minute
• The overall reaction for the conversion of substrate
to product can be written as follows:
E + S  ES  E + P
Example of an Enzyme Catalyzed Reaction
• The reaction for the sucrase catalyzed hydrolysis of
sucrose to glucose and fructose can be written as
follows:
E + S  ES  E + P1 + P2
where E = sucrase, S = sucrose, P1 = glucose and P2 =
fructose
Isoenzymes
• Isoenzymes are different forms of an enzyme that
catalyze the same reaction in different tissues in the
body
- they have slight variations in the amino acid
sequences of the subunits of their quaternary
structure
• For example, lactate dehydrogenase (LDH), which
converts lactate to pyruvate, consists of five
isoenzymes
Diagnostic Enzymes
• The levels of diagnostic enzymes in the blood can
be used to determine the amount of damage in
specific tissues
Temperature and Enzyme Activity
• Enzymes are most active at an optimum temperature
(usually 37°C in humans)
• They show little activity at low temperatures
• Activity is lost at high temperatures as denaturation
occurs
pH and Enzyme Activity
• Enzymes are most active at optimum pH
• Amino acids with acidic or basic side-chains have the
proper charges when the pH is optimum
• Activity is lost at low or high pH as tertiary structure is
disrupted
Optimum pH for Selected Enzymes
• Most enzymes of the body have an optimum pH of
about 7.4
• However, in certain organs, enzymes operate at lower
and higher optimum pH values
Enzyme Concentration and Reaction Rate
• The rate of reaction increases as enzyme concentration
increases (at constant substrate concentration)
• At higher enzyme concentrations, more enzymes are
available to catalyze the reaction (more reactions at
once)
• There is a linear relationship between reaction rate and
enzyme concentration (at constant substrate
concentration)
Substrate Concentration and Reaction Rate
• The rate of reaction increases as substrate
concentration increases (at constant enzyme
concentration)
• Maximum activity occurs when the enzyme is
saturated (when all enzymes are binding substrate)
• The relationship between reaction rate and substrate
concentration is exponential, and asymptotes (levels
off) when the enzyme is saturated
Enzyme Inhibitors
• Inhibitors (I) are molecules that cause a loss
of enzyme activity
• They prevent substrates from fitting into the
active site of the enzyme:
E + S  ES  E + P
E + I  EI  no P formed
Reversible Inhibitors (Competitive Inhibition)
• A reversible inhibitor
goes on and off, allowing
the enzyme to regain
activity when the inhibitor
leaves
• A competitive inhibitor
is reversible and has a
structure like the
substrate
- it competes with the
substrate for the active
site
- its effect is reversed by
increasing substrate
concentration
Example of a Competitive Inhibitor
• Malonate is a competitive inhibitor of succinate
dehydrogenase
- it has a structure that is similar to succinate
- inhibition can be reversed by adding succinate
Reversible Inhibitors (Noncompetitive Inhibition)
• A noncompetitive
inhibitor has a structure
that is different than that of
the substrate
- it binds to an allosteric
site rather than to the
active site
- it distorts the shape of the
enzyme, which alters the
shape of the active site and
prevents the binding of the
substrate
• The effect can not be
reversed by adding more
Irreversible Inhibitors
• An irreversible inhibitor destroys enzyme activity,
usually by bonding with side-chain groups in the active
site