Transcript Chem 7250 #1

Free energy of a reaction
The free energy change (DG) of a reaction determines
its spontaneity. A reaction is spontaneous if DG is
negative (if the free energy of products is less than that
of reactants).
F or a reaction A + B   C + D
o
D G = D G ' + R T ln
[C ] [D ]
[A ] [B ]
DGo' = standard free energy change (at pH 7, 1M
reactants & products); R = gas constant; T = temp.
F or a reaction A + B   C + D
D G = D G º' + R T ln
[C ] [D ]
[A ] [B ]
DGo' of a reaction may be positive, & DG negative,
depending on cellular concentrations of reactants and
products.
Many reactions for which DGo' is positive are
spontaneous because other reactions cause depletion of
products or maintenance of high substrate concentration.
At equilibrium
DG = 0.
K'eq, the ratio
[C][D]/[A][B] at
equilibrium, is the
equilibrium constant.
An equilibrium constant
(K'eq) greater than one
indicates a spontaneous
reaction (negative DG').
D G = D G º' + R T ln
[C ] [D ]
[A ] [B ]
   = D G º' + R T ln
D G º' = - R T ln
defining K ' eq =
[C ] [D ]
[A ] [B ]
[C ] [D ]
[A ] [B ]
[C ] [D ]
[A ] [B ]
D G º' = - R T ln K ' eq
DGo' = - RT ln K'eq
Variation of equilibrium constant with DGo‘ (25 oC)
K ' eq
D G º'
k J/m o l
S tartin g w ith 1 M reactan ts &
p ro du cts, th e reaction :
10
4
- 23
p ro ceed s forw ard (spo n tan eou s)
10
2
- 11
p ro ceed s forw ard (spo n tan eou s)
0
10 = 1
10
10
0
is at eq u ilib riu m
-2
+ 11
rev erses to form “reactan ts”
-4
+ 23
rev erses to form “reactan ts”
Energy coupling
 A spontaneous reaction may drive a non-spontaneous
reaction.
 Free energy changes of coupled reactions are additive.
A. Some 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.
For example, in the reaction catalyzed by the Glycolysis
enzyme Hexokinase, the half-reactions are:
ATP + H2O  ADP + Pi
DGo' = -31 kJ/mol
Pi + glucose  glucose-6-P + H2O
DGo' = +14 kJ/mol
Coupled reaction:
ATP + glucose  ADP + glucose-6-P DGo' = -17 kJ/mol
The structure of the enzyme active site, from which H2O
is excluded, prevents the individual hydrolytic reactions,
while favoring the coupled reaction.
B. Two separate reactions, occurring in the same cellular
compartment, 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  B + AMP + PPi
DGo' = + 15 kJ/mol
Enzyme 2:
PPi + H2O  2 Pi
DGo' = – 33 kJ/mol
Overall spontaneous reaction:
A + ATP + H2O  B + AMP + 2 Pi DGo' = – 18 kJ/mol
Pyrophosphate (PPi) is often the product of a reaction
that needs a driving force.
Its spontaneous hydrolysis, catalyzed by Pyrophosphatase
enzyme, drives the reaction for which PPi is a product.
Energy coupling in ion transport
Ion Transport may be
coupled to a chemical
reaction, e.g., hydrolysis or
synthesis of ATP.
In this diagram & below,
water is not shown. It should
be recalled that the ATP
hydrolysis/synthesis reaction
is: ATP + H2O  ADP + Pi.
AD P + Pi
S2
S1
ATP
S id e 1
S id e 2
S1
S2
S id e 1
S id e 2
The free energy change (electrochemical potential
difference) associated with transport of an ion S across
a membrane from side 1 to side 2 is:

D G = R T ln
[S ] 2
+ Z F DY
[S ] 1
R = gas constant, T = temperature, Z = charge on the ion,
F = Faraday constant, DY = voltage.
Since free energy changes
are additive, the
spontaneous direction
for the coupled reaction
will depend on relative
magnitudes of:
AD P + Pi
S2
S1
ATP
S id e 1
S id e 2
 DG for ion flux - varies with ion gradient & voltage.
 DG for chemical reaction - negative DGo' for ATP
hydrolysis; DG depends also on [ATP], [ADP], [Pi].
AD P + Pi
AD P + Pi
S2
S1
active
tran sp ort
ATP
H
+
H
1
ATP
syn th esis
+
2
ATP
Two examples:
Active Transport: Spontaneous ATP hydrolysis
(negative DG) is coupled to (drives) ion flux against a
gradient (positive DG).
ATP synthesis: Spontaneous H+ flux (negative DG) is
coupled to (drives) ATP synthesis (positive DG).
“High energy” bonds
NH
ATP
adenosine triphosphate
O
-O
P
O-
O
O
P
O-
N
N
O
O
P
O
N
N
adenine
CH 2
O
H
O-
phosphoanhydride
bonds (~ )
2
H
H
H
OH
OH
ribose
Phosphoanhydride bonds (formed by splitting out H2O
between 2 phosphoric acids or between carboxylic &
phosphoric acids) have a large negative DG of hydrolysis.
NH
ATP
adenosine triphosphate
O
-O
P
O-
O
O
P
adenine
N
O-
phosphoanh yd ride
bonds (~ )
P
N
N
O
O
2
O
CH
2
H
O-
N
O
ribose
H
H
H
OH
OH
Phosphoanhydride linkages are said to be "high energy"
bonds. Bond energy is not high, just DG of hydrolysis.
"High energy" bonds are represented by the "~" symbol.
~P represents a phosphate group with a large negative DG
of hydrolysis.
“High energy” bonds
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 group on glucose).
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)
 ATP often serves as an energy source.
Hydrolytic cleavage of one or both of the "high energy"
bonds of ATP is coupled to an energy-requiring
(non-spontaneous) reaction. (Examples presented earlier.)
 AMP functions as an energy sensor & regulator of
metabolism.
When ATP production does not keep up with needs, a
higher portion of a cell's adenine nucleotide pool is AMP.
AMP stimulates metabolic pathways that produce ATP.
• Some examples of this role involve direct allosteric
activation of pathway enzymes by AMP.
• Some regulatory effects of AMP are mediated by the
enzyme AMP-Activated Protein Kinase.
NH
Artificial ATP
analogs have
been designed
that are resistant
to cleavage of
the terminal
phosphate by
hydrolysis.
N
-O
O
H
O
P
N
P
O-
O-
N
O
O
P
O
CH
O-
2
H
OH
H
OH
H
N
N
O
H
A M P P N P (A D P N P ) A T P analog
Example: AMPPNP.
Such analogs have been used to study the dependence of
coupled reactions on ATP hydrolysis.
In addition, they have made it possible to crystallize an
enzyme that catalyzes ATP hydrolysis with an ATP
analog at the active site.
2
A reaction important for equilibrating ~P among
adenine nucleotides within a cell is that catalyzed by
Adenylate Kinase:
ATP + AMP  2 ADP
The Adenylate Kinase reaction is also important because
the substrate for ATP synthesis, e.g., by mitochondrial
ATP Synthase, is ADP, while some cellular reactions
dephosphorylate ATP all the way to AMP.
The enzyme Nucleoside Diphosphate Kinase (NuDiKi)
equilibrates ~P among the various nucleotides that are
needed, e.g., for synthesis of DNA & RNA.
NuDiKi catalyzes reversible reactions such as:
ATP + GDP  ADP + GTP,
ATP + UDP  ADP + UTP, etc.
Inorganic polyphosphate
Many organisms store energy as inorganic
polyphosphate, a chain of many phosphate residues
linked by phosphoanhydride bonds:
P~P~P~P~P...
Hydrolysis of Pi residues from polyphosphate may be
coupled to energy-dependent reactions.
Depending on the organism or cell type, inorganic
polyphosphate may have additional functions.
E.g., it may serve as a reservoir for Pi, a chelator of
metal ions, a buffer, or a regulator.
Why do phosphoanhydride linkages have a high DG
of hydrolysis? Contributing factors for ATP & PPi
include:
 Resonance stabilization of products of hydrolysis
exceeds resonance stabilization of the compound
itself.
 Electrostatic repulsion between negatively
charged phosphate oxygen atoms favors
separation of the phosphates.
Phosphocreatine (creatine
phosphate), another
compound with a "high
energy" phosphate linkage,
is used in nerve & muscle
for storage of ~P bonds.
O
-
O
CH3
H
N
P
O
-
C
NH2
N
O
CH2
C
+
O
-
phosphocreatine
Creatine Kinase catalyzes:
Phosphocreatine + ADP  ATP + creatine
This is a reversible reaction, though the equilibrium
constant slightly favors phosphocreatine formation.
 Phosphocreatine is produced when ATP levels are high.
 When ATP is depleted during exercise in muscle,
phosphate is transferred from phosphocreatine to ADP,
to replenish ATP.
O
O
-
O
C
C
CH2
PEP
ADP ATP
O PO 3
2-
O
+
O
O
-
C
C
C
H
-
OH
CH2
enolp yruvate
C
O
CH3
p yruvate
Phosphoenolpyruvate (PEP), involved in ATP synthesis
in Glycolysis, has a very high DG of Pi hydrolysis.
Removal of Pi from ester linkage in PEP is spontaneous
because the enol spontaneously converts to a ketone.
The ester linkage in PEP is an exception.
NH2
N
N
ester linkage
O
-O
P
O-
O
O
P
O-
N
O
O
P
O
CH2
O-
A T P (adenosine triphosphate)
adenine
O
H
H
OH
H
OH
H
N
ribose
Generally phosphate esters, formed by splitting out
water between a phosphoric acid and an OH group, have
a low but negative DG of hydrolysis. Examples:
 the linkage between the first phosphate and the ribose
hydroxyl of ATP.
O
6 CH
2
4
OH
P
OH
O
5
H
O
H
OH
3
H
OH
CH2
H
CH
O
1
H
2
HO
OH
CH2
O
P
O-
OH
OH
glu co se-6 -p h o sp h ate
O-
glycerol-3 -p h o sp h ate
Other examples of phosphate esters with low but
negative DG of hydrolysis:
 the linkage between phosphate & a hydroxyl group
in glucose-6-phosphate or glycerol-3-phosphate.
O
P rotein K inase
O H + ATP
P ro te in
P ro te in
O
P
O
Pi
O
-
+ ADP
-
H 2O
P rotein P hosphatase
 the linkage between phosphate and the hydroxyl
group of an amino acid residue in a protein (serine,
threonine or tyrosine).
Regulation of proteins by phosphorylation and
dephosphorylation will be discussed later.
ATP has special roles in energy coupling & Pi transfer.
DG of phosphate hydrolysis from ATP is intermediate
among examples below.
ATP can thus act as a Pi donor, & ATP can be synthesized
by Pi transfer, e.g., from PEP.
D G ' o f p h o sp h ate
h y d ro ly sis, k J /m o l
o
C o m p ou n d
P h o sp h o en o lp y ru va te (P E P )
-  
P h o sp h o creatin e
-  
P y ro p h o sp h ate
-  
A T P (to A D P )
-  
G lu co se-6-p h osp h ate
-  
G ly cero l-3-p h osp h ate
-  
O
Some other
“high energy”
bonds:
C o e n zym e A -S H
+
HO
C
R
O
C o e n zym e A - S
C
R
+ H2O
A thioester forms between a carboxylic acid & a thiol
(SH), e.g., the thiol of coenzyme A (abbreviated CoA-SH).
Thioesters are ~ linkages. In contrast to phosphate esters,
thioesters have a large negative DG of hydrolysis.
O
C o e n zym e A -S H
+ HO
C
CH3
acetic acid
O
C o e n zym e A - S
C
C H 3 + H 2O
acetyl-C oA
The thiol of coenzyme A can react with a carboxyl group
of acetic acid (yielding acetyl-CoA) or a fatty acid
(yielding fatty acyl-CoA).
The spontaneity of thioester cleavage is essential to the
role of coenzyme A as an acyl group carrier.
Like ATP, CoA has a high group transfer potential.
SH
CH 2
Coenzyme A includes
b-mercaptoethylamine,
in amide linkage to the
carboxyl group of the B
vitamin pantothenate.
The hydroxyl of
pantothenate is in ester
linkage to a phosphate
of ADP-3'-phosphate.
The functional group is
the thiol (SH) of
b-mercaptoethylamine.
b -m ercap to ethylam ine
CH 2
NH
C
O
CH 2
p anto thenate
CH 2
NH
C
NH 2
O
HO
C
H
H 3C
C
CH 3 O
H 2C
O
N
N
P
O-
O
O
P
N
N
O
CH 2
O-
O
H
H
O
OH
H
A D P -3 '-p ho sp hate
-
C oenzym e A
O
H
P
O
O
-
3',5'-Cyclic AMP (cAMP), is used
by cells as a transient signal.
Adenylate Cyclase catalyzes cAMP
synthesis: ATP  cAMP + PPi.
The reaction is highly spontaneous
due to the production of PPi, which
spontaneously hydrolyzes.
NH2
cA M P
N
N
N
N
H2
4'
5'C
O
H
O
H
3'
H
H
OH
1'
2'
P
O
Phosphodiesterase catalyzes
O
hydrolytic cleavage of one Pi ester
O(red), converting cAMP  5'-AMP.
This is a highly spontaneous reaction, because cAMP is
sterically constrained by having a phosphate with ester
links to 2 hydroxyls of the same ribose. The lability of
cAMP to hydrolysis makes it an excellent transient signal.
List compounds exemplifying the following roles
of "high energy" bonds:
 Energy transfer or storage
ATP, PPi, polyphosphate, phosphocreatine
 Group transfer
ATP, Coenzyme A
 Transient signal
cyclic AMP
Kinetics vs Thermodynamics:
A high activation energy barrier usually causes
hydrolysis of a “high energy” bond to be very slow in
the absence of an enzyme catalyst.
This kinetic stability is essential to the role of ATP and
other compounds with ~ bonds.
If ATP would rapidly hydrolyze in the absence of a
catalyst, it could not serve its important roles in energy
metabolism and phosphate transfer.
Phosphate is removed from ATP only when the reaction
is coupled via enzyme catalysis to some other reaction
useful to the cell, such as transport of an ion,
phosphorylation of glucose, or regulation of an enzyme
by phosphorylation of a serine residue.
Oxidation & reduction
Oxidation & reduction will be covered in more detail later.
The evolution of photosynthesis, and the generation of
the oxygen that is now plentiful in our environment,
allowed development of metabolic pathways that derive
energy from transfer of electrons from various reductants
ultimately to molecular oxygen.
Oxidation of an iron atom involves loss of an electron
(to an acceptor): Fe++ (reduced)  Fe+++ (oxidized) + eSince electrons in a C-O bond are associated more with O,
increased oxidation of a C atom means increased number
of C-O bonds.
H
H
C
H
H
H
C
OH
H
H
O
O
O
C
C
C
H
H
H
OH
O
In c re a s in g o x id a tio n o f c a rb o n
Oxidation of carbon is spontaneous (energy-yielding).
Two important e- carriers in metabolism: NAD+ & FAD.
NAD+, Nicotinamide
Adenine Dinucleotide,
is an electron acceptor
in catabolic pathways.
The nicotinamide ring,
derived from the vitamin
niacin, accepts 2 e- & 1
H+ (a hydride) in going
to the reduced state,
NADH.
NADP+/NADPH is
similar except for Pi.
NADPH is e- donor in
synthetic pathways.
N icotinam ide
A denine
D inucleotide
H
C
NH
O
-
O
P
+
N
O
CH
2
O
H
OH
NH
O
2
N
N
P
O
CH
O
nicotinam ide
H
OH
O
2
H
H
-
O
N
2
H
N
O
H
H
H
OH
adenine
OH
esterified to
+
P i in N A D P
NAD+/NADH
H
O
H
H
C
C
NH 2
+
N
2e
R
NAD
O
NH 2
-
+ H
+
N
R
+
NADH
The electron transfer reaction may be summarized as :
NAD+ + 2e- + H+  NADH.
It may also be written as:
NAD+ + 2e- + 2H+  NADH + H+
dim ethylisoalloxazine
O
H
C
C
N
O
-
H 3C
C
C
C
NH
H 3C
C
C
C
C
C
H
N
2e + 2H
H
C
+
O
N
H
N
H 3C
C
C
C
NH
H 3C
C
C
C
C
C
H
CH2
FA D
N
O
N
H
CH2
HC
OH
HC
OH
HC
OH O
H 2C
C
O
P
O-
A d e n in e
O
O
P
O-
O
R ib o se
FA D H 2
HC
OH
HC
OH
HC
OH O
H 2C
O
P
O-
A d e n in e
O
O
P
O
O-
FAD (Flavin Adenine Dinucleotide), derived from the
vitamin riboflavin, functions as an e- acceptor.
The dimethylisoalloxazine ring undergoes
reduction/oxidation.
FAD accepts 2 e- + 2 H+ in going to its reduced state:
FAD + 2 e- + 2 H+  FADH2
R ib o se
 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.