Biochemical Energetics
Transcript of Biochemical Energetics
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Biochemical Energetics
Copyright 1999-2004 by Joyce J. Diwan.
All rights reserved.
Biochemistry of Metabolism
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Free energy of a reaction
The free energy change ((G) of a reaction determines
its spontaneity. A reaction is spontaneous if(G is
negative (if the free energy of products is less than that
of reactants).
(Go' = standard free energy change (at pH 7, 1M
reactants & products); R= gas constant;T = temp.
For a reaction A + B C + D
(G = (Go
' + RT ln[C][D]
[A][B]
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(Go'of a reaction may be positive, & (G negative,
depending on cellular concentrations of reactants and
products.
Many reactions for which (Go' is positive arespontaneous because other reactions cause depletion of
products or maintenance of high substrate concentration.
For a reaction
G G' RT l[A][ ]
[ ][ ]
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At equilibrium
(G = 0.
K'eq, the ratio
[C][D]/[A][B] atequilibrium, is the
equilibrium constant.
An equilibrium constant(K'eq) greater than one
indicates a spontaneous
reaction (negative (Gr').
(G (G' RT l
(G' RT l
(G' -RTl
de ining K'e
(G' -RT l K 'e
[C][ ][A][ ]
[C][ ]
[A][ ]
[C][ ][A][ ]
[C][ ]
[A][ ]
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K'e(G
'
kJ/mol
Starting ith 1 reactants
products, the reaction:
104
- 23 proceeds or ar d (spontaneous)
102
-11 proceeds or ar d (spontaneous)
100
1 0 is at e ili ri m
10-2 11 reverses to orm reactants
10-4 + 23 reverses to orm reactants
(Go' = RT ln K'eq
Variation of equilibrium constant with (Go (25 oC)
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Energy coupling
A spontaneous reaction may drive a non-spontaneous
reaction.
Free energy changes of coupled reactions are additive.
A. ome enzyme-catalyzed reactions are interpretable as
two coupled half-reactions, one spontaneous and the
other non-spontaneous.
At the enzyme active site, the coupled reaction is
kinetically facilitated, while individual half-reactions
are prevented.
Free energy changes of half reactions may be summed,
to yield the free energy of the coupled reaction.
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For example, in the reaction catalyzed by the Glycolysis
enzyme Hexokinase, the half-reactions are:
ATP + H2O m ADP + Pi (Go' =31 kJ/mol
Pi + glucosem glucose-6-P + H2O (Go' = +14 kJ/mol
Coupled reaction:
ATP + glucose mADP + glucose-6-P (Go' =17 kJ/mol
The structure of the enzyme active site, from which H2Ois excluded, prevents the individual hydrolytic reactions,
while favoring the coupled reaction.
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B. Two separate reactions, occurring in the same cellularcompartment, one spontaneous and the other not, may be
coupled by a common intermediate (reactant or product).A hypothetical, but typical, example involving PPi:
Enzyme 1:
A + ATP m B + AMP + PPi (Go' = + 15 kJ/mol
Enzyme 2:PPi + H2O m 2 Pi (G
o' = 33 kJ/mol
Overall spontaneous reaction:
A + ATP + H2O m B + AMP + 2 Pi (Go' = 18 kJ/mol
Pyrophosphate (PPi) is often the product of a reactionthat needs a driving force.
Its spontaneous hydrolysis, catalyzed by Pyrophosphataseenzyme, drives the reaction for which PPi is a product.
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Energy coupling in ion transport
Ion Transport may be
coupled to a chemical
reaction, e.g., hydrolysis orsynthesis of ATP.
In this diagram & below,
water is not shown. It should
be recalled that the ATP
hydrolysis/synthesis reaction
is: ATP + H2Om ADP + Pi.
S1 S2
ATP
ADP + Pi
Si e 1 Si e 2
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The free energy change (electrochemical potential
difference) associated with transport of an ionS
acrossa membrane from side 1 to side 2 is:
R= gas constant, T = temperature, Z = charge on the ion,
F =Faraday constant, (= = voltage.
(G R T l + Z F (=
[S]1
[S]2
S1 S2
Side 1 Side 2
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(G for ion flux - varies with ion gradient & voltage. (G for chemical reaction - negative(Go' for ATP
hydrolysis;(G depends also on [ATP], [ADP], [Pi].
ince free energy changes
are additive, the
spontaneous direction
for the coupled reactionwill depend on relative
magnitudes of:
S1 S2
ATP
ADP + Pi
Side 1 Side 2
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Two examples:
Active Transport: pontaneous ATP hydrolysis
(negative(
G) is coupled to (drives) ion flux against a
gradient (positive (G).
ATPsynthesis: pontaneous H+ flux (negative (G) is
coupled to (drives) ATP synthesis (positive (G).
S1 S2
ATP
ADP + Pi
active
tra sport
H+
1 H+
2
ATP
ADP + Pi
ATP
sy thesis
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N
NN
N
NH2
O
OHOH
HH
H
CH2
H
OPOPOP-O
O
O- O-
O O
O-
adenine
ribose
ATP
adenosine triphosphate
phosphoanhydride
bonds (~)
Phosphoanhydride linkages are said to be "high energy"
bonds. Bond energy is not high, just (G of hydrolysis.
"High energy" bonds are represented by the "~" symbol.
~P represents a phosphate group with a large negative (G
of hydrolysis.
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Compounds with high energy bonds are said to
have high group transfer potential.
For example, Pi may be spontaneously cleaved from
ATP for transfer to another compound, e.g., to a
hydroxyl on glycerol.
High energy bonds
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Potentially, 2 ~P bonds can be cleaved, as 2 phosphates
are released by hydrolysis from ATP.
AMP~P~P AMP~P + Pi (ATP ADP + Pi)
AMP~P AMP + Pi (ADP AMP + Pi)
Alternatively:
AMP~P~P AMP + P~P (ATP AMP + PPi)
P~P 2 Pi (PPi 2Pi)
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Example: AMPPNP.
uch analogs have been used to study the dependence of
coupled reactions on ATP hydrolysis.
In addition, they have made it possible to crystallize anenzyme that catalyzes ATP hydrolysis with an ATPanalog at the active site.
AMPPNP (ADPNP) ATP analog
N
NN
N
NH2
O
OHOH
HH
H
CH2
H
OPOPNP-O
O
O- O-
O O
O-
H
Artificial ATPanalogs have
been designedthat are resistantto cleavage ofthe terminal
phosphate byhydrolysis.
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Inorganic polyphosphate
Many organisms store energy as inorganicpolyphosphate, a chain of many phosphate residueslinked by phosphoanhydride bonds:
P~P~P~P~P...
Hydrolysis of Pi residues from polyphosphate may becoupled to energy-dependent reactions.
Depending on the organism or cell type, inorganicpolyphosphate may have additional functions.
E.g., it may serve as a reservoir for Pi, a chelator ofmetal ions, a buffer, or a regulator.
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Why do phosphoanhydride linkages have a high (G
of hydrolysis? Contributing factors for ATP & PPiinclude:
Resonance stabilization of products of hydrolysis
exceeds resonance stabilization of the compound
itself.
Electrostatic repulsionbetween negativelycharged phosphate oxygen atoms favors
separation of the phosphates.
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Creatine Kinase catalyzes the reversible reaction:
Phosphocreatine + ADPm ATP + creatine
Phosphocreatine is produced when ATP levels are high.
During exercise in muscle, phosphate is transferred fromphosphocreatine to ADP, to replenish ATP.
Phosphocreatine may also be used to transport ~P fromone compartment of a cell to another.
O P
H
N C
O
O
N
NH2+
CH2
CH3
C
O
O
phosphocreatine
Phosphocreatine (creatinephosphate), anothercompound with a "highenergy" phosphate linkage,is used in nerve & muscleforstorage of ~P bonds.
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A reaction important for equilibrating ~P amongadenine nucleotides within a cell is that catalyzed by
Adenylate Kinase:ATP + AMPm 2 ADP
The Adenylate Kinase reaction is also important becausethe substrate for ATP synthesis, e.g., by mitochondrial
ATP ynthase, is ADP, while some cellular reactionsdephosphorylate ATP all the way to AMP.
The enzyme Nucleoside Diphosphate Kinase (NuDiKi)equilibrates ~P among the various nucleotides that areneeded, e.g., for synthesis of DNA & RNA.
NuDiKi catalyzes reversible reactions such as:
ATP + GDPm ADP + GTP,
ATP + UDPm ADP + UTP, etc.
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Phosphoenolpyruvate (PEP), involved in ATP synthesisin Glycolysis, has a very high (G of Pi hydrolysis.
Removal of Pi from ester linkage in PEP is spontaneousbecause the enol spontaneously converts to a ketone.
The ester linkage in PEP is an exception.
C
C
O O
OPO32
CH2
C
C
O O
O
CH3
C
C
O O
OH
CH2
ADP ATP
H+
PEP enolpyruvate pyruvate
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Generally phosphate esters (formed by splitting out
water between a phosphoric acid and an OH group) havea low but negative (Gof hydrolysis. Examples:
the linkage between the first phosphate of ATP & theribose hydroxyl
N
NN
N
NH2
O
OHOH
HH
H
CH2
H
OPOPOP-O
O
O- O-
O O
O-
adenine
ribose
ATP(adenosine triphosphate)
ester linkage
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Other examples ofphosphate esters with low but
negative (G of hydrolysis: the linkage between phosphate & a hydroxyl group
in glucose-6-phosphate orglycerol-3-phosphate.
glycerol-3-phosphate
CH2
CH
CH2
OH
HO
O P
O
O
O
H O
OH
H
OHH
OH
CH2
H
OH
H
1
6
5
4
3 2
O P
O
OH
OH
glucose-6-phosphate
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ATP has special roles in energy coupling & Pi transfer.
(G of phosphate hydrolysis from ATP is intermediate
among examples below.ATP can thus act as a Pi donor, & ATP can be synthesizedby Pi transfer, e.g., from PEP.
Compound(G
o'of phosphate
hydrolysis, kJ/mol
Phosphoenolpyruvate (PEP)
Phosphocreatine
Pyrophosphate
ATP (to ADP)
Glucose-6-phosphate
Glycerol-3-phosphate
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Coenzyme A-SH + HO C
O
R
Coenzyme A-S C
O
R + H2O
A thioester forms between a carboxylic acid & a thiol
( H), e.g., the thiol ofcoenzyme A (abbreviated CoA- H).
Thioesters are ~ linkages. In contrast to phosphate esters,
thioesters have a large negative (G of hydrolysis.
ome otherhigh energy
bonds:
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The thiol of coenzyme A can react with a carboxyl groupof acetic acid (yielding acetyl-CoA) or a fatty acid
(yielding fatty acyl-CoA).The spontaneity of thioester cleavage is essential to therole of coenzyme A as an acyl group carrier.
Like ATP, CoA has a high group transfer potential.
Coenz e - H + HO C
O
CH3
Coenz e - C
O
CH3 + H2O
acetic acid
acetyl-CoA
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Coenzyme A includes
F-mercaptoethylamine,in amide linkage to the
carboxyl group of the B
vitamin pantothenate.
The hydroxyl ofpantothenate is in ester
linkage to a phosphate
ofADP-3'-phosphate.
The functional group is
the thiol ( H) of
F-mercaptoethylamine.
N
NN
N
NH2
O
OHO
HH
H
CH2
H
OPOPOH2C
O
O O
O
P
O
O
O
C
C
C
NH
CH2
CH2
C
NH
CH3H3C
HHO
O
CH2
CH2
SH
O
F-mercaptoethylamine
pantothenate
P-3 -phosphate
oenzyme
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3',5'-Cyclic AMP (cAMP), is usedby cells as a transient signal.
Adenylate Cyclase catalyzes cAMPsynthesis: ATPp cAMP + PPi.
The reaction is highly spontaneous
due to the production ofPPi, whichspontaneously hydrolyzes.
Phosphodiesterase catalyzeshydrolytic cleavage of one Pi ester(red), converting cAMPp 5'-AMP.
N
NN
N
NH2
O
OHO
HH
H
H2C
HO
PO
O-
1'
3'
5' 4'
2'
cAMP
This is a highly spontaneous reaction, because cAMP issterically constrained by having a phosphate with esterlinks to 2 hydroxyls of the same ribose. The lability ofcAMP to hydrolysis makes it an excellent transient signal.
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List compounds exemplifying the following rolesof "high energy" bonds:
Energy transfer or storage
ATP, PPi, polyphosphate, phosphocreatine
Group transfer
ATP, Coenzyme A
Transient signal
cyclic AMP
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Oxidation & reduction
Oxidation of an iron atom involves loss of an electron
(to an acceptor): Fe++ (reduced) Fe+++ (oxidized) + e-
Since electrons in a C-O bond are associated more with
O, increased oxidation of a C atom means increased
number of C-O bonds. Oxidation of C is spontaneous.
Increasing oxidation of carbon
H
CH H
H
H
CH OH
H
H
C
H
O
O
C
O
OH
C
H
O
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NAD+, NicotinamideAdenine Dinucleotide,
is an electronacceptorin catabolic pathways.
The nicotinamide ring,derived from the
vitamin niacin, accepts2 e- & 1 H+ (a hydride)in going to the reducedstate, NADH.
NADP+/NADPH issimilar except for Pi.NADPH is e donor insynthetic pathways.
H
C NH2
O
CH2
H
N
HOH OH
H H
OOP
O
HH
OH OH
H H
OCH2
N
N
N
NH2
OP
O
O
O
+
NO
nicotinamide
adenine
esterified toPi in NADP
+
Nicotinamide
AdenineDinucleotide
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NAD+/NADH
The electron transfer reaction may be summarized as :NAD+ + 2e + H+m NADH.
It may also be written as:
NAD+ + 2e + 2H+m NADH + H+
N
R
H
C
NH2
O
N
R
C
NH2
OH H
+
2
e
+
H+
NAD+
NADH
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FAD (Flavin Adenine Dinucleotide), derived from the
vitamin riboflavin, functions as an e acceptor. Thedimethylisoalloxazine ring undergoes reduction/oxidation.
FAD accepts 2 e- + 2 H+ in going to its reduced state:
FAD + 2 e- + 2 H+m FADH2
C
CC
H
C
C
H
C
NC
C
N
NC
NH
C
H3C
H3C
O
O
CH2
HC
HC
HC
H2C
OH
O P O P O
O
O-
O
O-
Ribose
OH
OH
Adenine
C
CC
H
C
C
H
C
NC
C
H
N
N
H
C
NH
C
H3C
H3C
O
O
CH2
HC
HC
HC
H2C
OH
O P O P O
O
O-
O
O-
Ribose
OH
OH
AdenineFAD FADH2
2 e + 2 H+
dimethylisoalloxazine
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NAD+ is a coenzyme, that reversibly binds to
enzymes.
FAD is a prosthetic group, that remains tightly
bound at the active site of an enzyme.