Thermodynamics & ATP
Review thermodynamics,
energetics, chemical
sense, and role of ATP
Lecture 24
Thermodynamics in Biology
A Simple Thought Experiment
48 hours
1 E. coli cell (10 -11 mL)
1 mL H 2O
5 mg glucose
1 mg (NH 4)SO4
Mg++, PO4=, Fe3+, etc...
Glucose + (NH 4)SO4
109 cells
1 mL H 2O
0 mg glucose
<1 mg (NH 4)SO4
CO2
Cells + CO 2
Driving Forces for Natural Processes
• Enthalpy
– Tendency toward lowest energy state
• Form stablest bonds
• Entropy
– Tendency to maximize randomness
Enthalpy and Bond Strength
• Enthalpy = ∆H = heat change at constant pressure
• Units
– cal/mole or joule/mole
• 1 cal = 4.18 joule
• Sign
– ∆H is negative for a reaction that liberates heat
Entropy and Randomness
Decreased
randomness
Myoglobin
153 free
amino acids
Entropy and Randomness
• Entropy = S = measure of randomness
– cal/deg·mole
• T∆S = change of randomness
• For increased randomness, sign is “+”
“System” Definition
System
Surroundings
Closed system:
No exchange of
mass or energy
“System” Definition
Isolated system:
Energy is exchanged
E
E
“System” Definition
M
Open System:
Mass and energy
are exchanged
E
M
E
Cells and Organisms: Open Systems
• Material exchange with surroundings
– Fuels and nutrients in (glucose)
– By-products out (CO2)
• Energy exchange
– Heat release (fermentation)
– Light release (fireflies)
– Light absorption (plants)
1st Law of Thermodynamics
• Energy is conserved, but transduction is allowed
• Transduction
One form
of E
Light
Another form
of E
Plants
Mayer: 1842
Chemical
bonds
2nd Law of Thermodynamics
• In all spontaneous processes, total entropy
of the universe increases
2nd Law of Thermodynamics
• ∆Ssystem + ∆Ssurroundings = ∆Suniverse > 0
• A cell (system) can decrease in entropy only
if a greater increase in entropy occurs in
surroundings
• C6H12O6 + 6O2  6CO2 + 6H2O
complex
simple
Entropy: A More Rigorous Definition
• From statistical mechanics:
– S = k lnW
• k = Boltzmann constant = 1.3810–23 J/K
• W = number of ways to arrange the
system
• S = 0 at absolute zero (-273ºC)
Gibbs Free Energy
• Unifies 1st and 2nd laws
• ∆G
– Gibbs free energy
– Useful work available in a process
• ∆G = ∆H – T∆S
– ∆H from 1st law
• Kind and number of bonds
– T∆S from 2nd law
• Order of the system
∆G
• Driving force on a reaction
• Work available  distance from equilibrium
• ∆G = ∆H – T∆S
– State functions
•
•
•
•
Particular reaction
T
P
Concentration (activity) of reactants and products
Equilibrium
• ∆G = ∆H – T∆S = 0
• So ∆H = T∆S
– ∆H is measurement of enthalpy
– T∆S is measurement of entropy
• Enthalpy and entropy are exactly balanced at
equilibrium
Effects of ∆H and ∆S on ∆G
Voet, Voet, and Pratt. Fundamentals of Biochemistry. 1999.
Standard State and ∆Gº
• Arbitrary definition, like sea level
• [Reactants] and [Products]
– 1 M or 1 atmos (activity)
• T = 25ºC = 298K
• P = 1 atmosphere
• Standard free energy change = ∆Gº
Biochemical Conventions: ∆Gº
• Most reactions at pH 7 in H2O
• Simplify ∆Gº and Keq by defining [H+] = 10–7 M
• [H2O] = unity
• Biochemists use ∆Gº and Keq
Relationship of ∆G to ∆Gº
• ∆G is real and ∆Gº is standard
• For A in solution
– GA = GA + RT ln[A]
}
º
• For reaction aA + bB  cC + dD
[C]c [D]d
– ∆G = ∆Gº + RT ln
[A]a [B]b
– Constant Variable
(from table)
Relationship Between ∆Gº and Keq
[C]c [D]d
• ∆G = ∆Gº + RT ln a
[A] [B]b
• At equilibrium, ∆G = 0, so
[C]c [D]d
– ∆Gº = –RT ln
[A]a [B]b
– ∆Gº = –RT ln Keq
Relationship Between Keq and ∆Gº
Keq ²G º (kJ/mol)
10-6
34.3
10-5
28.5
10-4
21.4
-3
10
17.2
-2
10
11.3
10-1
5.9
1
0.0
1
10
-5.9
102
-11.3
103
-17.2
Will Reaction Occur Spontaneously?
A+B
C+D
• When:
– ∆G is negative, forward reaction tends to occur
– ∆G is positive, back reaction tends to occur
– ∆G is zero, system is at equilibrium
c [D]d
[C]
∆G = ∆Gº + RT ln
[A]a [B]b
A Caution About ∆Gº
• Even when a reaction has a large, negative
∆Gº, it may not occur at a measurable rate
• Thermodynamics
– Where is the equilibrium point?
• Kinetics
– How fast is equilibrium approached?
• Enzymes change rate of reactions, but do
not change Keq
∆Gº is Additive (State Function)
Reaction
AB
BC
Sum: A  C
Free energy change
∆G1º
∆G2º
∆G1º + ∆G2º
Also: B  A
– ∆G1º
Coupling Reactions
∆Gº
kcal/mol kJ/mol
Glucose + HPO42–  Glucose-6-P
+3.3
+13.8
ATP
 ADP + HPO42–
–7.3
–
ATP + Glucose
 ADP + Glucose-6-P 30.5
–4.0
–
16.7
Resonance Forms of Pi
O
HO
P
O
O
HO
O
P
O
–
O
O
– O
O
HO
P
O
O
–
O
O
HO
P
P
O
O
So: resonance stabilization
etc...
O –
Phosphate Esters and Anhydrides
Esters:
H2O
O
ROH + HO
P
O
O
R
O
O
P
O
O
H2O
Anhydrides:
O
R
C
H2O
O
+ HO
OH
P
O
O
R
C
O
O
O
P
O
H2O
= Hydrolysis
O
Hydrolysis of Glucose-6-Phosphate
O
HO
P
O
O
HO
CH2
O + H2O
O
CH2
O
+ HO
P
O
∆Gº = –3.3 kcal/mol
= –13.8 kJ/mol
Ionization,
resonance
Product
stabilization
OH
High ∆Gº Hydrolysis Compounds
O
O
C
C
O
H2O
O
O
CH2
P
Phospho-enol
pyruvate
(PEP)
O
C
O
O
O
C
Pi
O
C
OH
CH2
C
O
CH3
enol-keto
isomerization
∆Gº = –14.8 kcal/mol = –61.9 kJ/mol
High ∆Gº Hydrolysis Compounds
O
O
P
O
O
O
C
HO
O
CH
CH2
O
H2O
O
O
P
P
O
CH2
O
O
Pi H
C
O
O
1,3-Bisphosphoglyceric Acid
(1,3-diPGA)
CH OH
O
Resonance
stabilized
∆Gº = –11.8 kcal/mol = –49.3 kJ/mol
High ∆Gº Hydrolysis Compounds
O
O
C
O
C
CH2
H2O
N
CH3
CH2
C
NH
N
CH3
C
NH
NH
O
O
P
O
O
Phosphocreatine
+ Pi
NH 2
Resonance
possible
∆Gº = –10.3 kcal/mol = –43 kJ/mol
Phosphate Anhydrides (Pyrophosphates)
O
O
P
O
O
O
P
O
O
O
Repulsion
P
O
O
CH2
O
HO
Adenosine triphosphate
(ATP)
Adenine
OH
H2O
ADP + P i
∆Gº = –7.3 kcal/mol = –30.5 kJ/mol
Thiol Esters
H2O
O
CoA
S
C
CH3
CoASH
+
H3C
O
C
+
H
∆Gº = –7.5 kcal/mol = –31.4 kJ/mol
O
Thiol Esters
O
O
C
R1
S
R2
C
R1
O
O
C
R1
S
R2
O
R2
C
R1
O
R2
Thiol ester less resonance-stabilized
“High-Energy” Compounds
• Large ∆Gº hydrolysis
– Bond strain (electrostatic repulsion) in reactant
• ATP
– Products stabilized by ionization
• Acyl-P
– Products stabilized by isomerization
• PEP
– Products stabilized by resonance
• Creatine-P
“High-Energy” Compounds
• “High-energy” compound is one with a
∆Gº below –6 kcal/mol (–25 kJ/mol)
High-Energy Compounds
Group Transfer Potential
Lecture 25
Chemical Sense in Metabolism
Making and Breaking C–C
Bonds
• Homolytic reactions
AB
A +B
• Heterolytic reactions
AB
A+ B
Making and Breaking C–C
Bonds
• Nucleophilic substitutions
R W+ Z
Leaving Nucleophile
group
RZ+ W
•
Nucleophilic Substitution
Reactions
SN1
H
a) R W
R +W
HW
b) R + Z
Carbocation
RZ
Carbocation
Stability
o
o
o
CH 3 < 1 < 2 < 3
Reactivity
o
o
o
3 > 2 > 1 > CH 3
R1
R2
C
R3
(Planar)
Racemization
Common Biological
Nucleophiles
SN2 Nucleophilic Substitution
Z
R1 R2
+ C W
R3
R1
–
Z
C
R2
W
–
R3
R2 R
1
Z
C
+ W
R3
Reactivity is SN2 Reactions
Nucleophile
–
–
–
I , HS ,RS
Br–,HO–,RO–,CN–
NH3;Cl–,RCOO–
H2O, ROH
RCOOH
Stronger
nucleophilic RO
base
Reactivity
Very good
Good
Fair
Weak
Very weak
O
> R C
O
5
>10
104
103; 101-102
1
-2
10
Weaker
nucleophilic
base
Leaving Group
• Must accommodate a pair of electrons
– And sometimes a negative charge
Major Role of Phosphorylation
• Converts a poor leaving group (–OH) into a
good one (Pi, PPi)
PPi > Pi > H2O >
OH
Acid Catalysis of Substitution
Reactions
Poor
ROH
R + OH
leaving group
ROH2
H
ROH
Good
+
R
H2O
leaving group
This H is often donated by an
acidic sidechain of enzyme
Central Importance of
Carbonyls
1. Can produce a carbocation
O
O
C
C
2. Can stabilize a carbanion
O
O
C
C
C
C
Biological Carbonyls
NH 3
R
C
O
Amino
acids
C
O
O
H3C
Fatty
acids
(CH2)n C
O
O
R
CH2 C
O
-keto acyl
(fat oxidation
and synthesis)
COO
-ketoglutarate
(Krebs cycle)
O
CH2 C
O
OOC
CH2
CH2
C
Aldol Condensation
R1
H
C
R2
R1
O
C
C
R3
H
R2
O
C
R3
Aldol Condensation
R4
O
R1
C
R5
C
O
C
R3
R2
R4 R1
O
C
C
R5 R2
O
C
R3
Aldol Condensation
H
R4 R1
O
C
C
R5 R2
R4 R1
O
C
HO
R3
C
C
R5 R2
O
C
R3
Aldolase Reaction
• Glycolysis and gluconeogenesis
Glyceraldehyde- Dihydroxyacetone3-phosphate
phosphate
R4
O
R1
+ H
C
R5
C
R2
Aldolase
R4 R1
HO
C
C
R5 R2
O
C
R3
Fructose-1,6bisphosphate
O
C
R3
Claisen Condensation
carbanion + ester carbonyl
O
R1
C
R2
R5
C
R4
+
C
O
R3
O
R1
ketone
O
R5 R2
C
C
C
R4 O
R3
O
Claisen Condensation
R1
O
R5 R2
C
C
C
O
R1
R4 O
O
R5 R2
C
C
C
R4
+
R3
H
HO
R3
O
R3
O
Thioesters in Biology
O
R1
C
Oxygen ester
O
R2
O
R1
C
Thioester
S
R2
• In thioesters, the carbonyl carbon has more
positive character than carbonyl carbon in
oxygen ester.
“High-Energy” Thioester
Compounds
Coenzyme A
SH
H
C
H -mercapto-
H
C
H
HN
C
O
ethylamine
H
H
C
C
H
H
N
H
H
CH3 H
C
C
C
O
OH CH3 H
C
O
O
P
O
ADP
O
O
P
O
O
CH2 Adenine
O
Pantothenic acid
(growth factor)
O
O
P
O
OH
O
Fatty Acid Metabolism
• Uses Claisen condensation
SCoA
O
O
C
CH2
R
C
H2C
SCoA
Thiolase
O
R
CH2
C
O
CH2
C
SCoA
+
CoASH
• Thiolase acts in fatty acid oxidation for
energy production
Thiolase: Role of Cys-SH
O
Enz
SH
+ R C
S
CoA
O
Enz
S
C
R
+ CoASH
O
R1
C
Enz
CH2
SH +
R
O
O
C
CH2 C
R1
Thiolase: Role of Cys-SH
H
Enz
SH
+I
C
H
O
C
HI
H
Enz
O
S
C
H
O
C
O
Energy Diagram for Reaction
• ‡ is the transition state
– Pentacovalent carbon, for example
Functional Groups on
Enzymes
• Amino acid side chains
–
SH
–
O
C
– Imidazole
–
CH2OH
O
Functional Groups on
Enzymes
• Coenzymes/cofactors
– Pyridoxal phosphate
• Metal ions and complexes
– Mg2+, Mn2+, Co2+, Fe2+, Zn2+, Cu2+, Mo3+
Enzyme Inhibitors and
Poisons
•
SH + Hg 2+
(heavy metals)
• Chelating agents
– EDTA (divalent cations)
– CN– (Fe2+)
• Cofactor analogs
– Warfarin
• Suicide substrates
S
Hg
Lecture 26
ATP and Phosphoryl Group
Transfers
Phosphate Esters and
Anhydrides
O
OH
O
P
O
O
O
P
O
R2O
OR
OR
Diester
O
ribose
Anhydride
O
OR1
O
Monoester
Adenine
P
P
O
O
O
P
O
Mg 2+
O
O
P
O
O
Phosphoryl Group Transfers
PO43- has
nucleophilic O – O
+
O P is electrophile
HO
P
OH
O
OH
P
O
O
pK3=12.7
O
HO
P
OH
O
OH
pK1=~1
HO
P
O
OH
pK2=6.7
O
O
P
O
OH
Phosphoryl (Not Phosphate)
Transfers
Glucose-1-phosphate
CH2OH
O
OH
OH
O
OH
18
O
P
O
CH2OH
O
OH
OH
OH
+ OH
O
O
O -labeled
O
H
H
(M. Cohn)
HO
P
O
O
Nucleophilic Displacements
OR1
R2O
R2O
R3O
P
OR3
OR1
O
P
OR4
O
(nucleophile)
H
O
R4
OR1
R3O P
R4O
+
R2OH
O
ATP as a Phophoryl Donor
• 2 roles for ATP
– Thermodynamic
• Drive unfavorable reactions
– Mechanistic
• Offer 3 electrophilic
phosphorous atoms for
nucleophilic attack
ATP as Phosphoryl Donor
• 3 points of nucleophilic attack
O


O
O
P
O
O
P
O

O
O
P
O
Ribose
Adenine
O
PhosphorPyrophos- Adenylation (AMP)
ylation phorylation
Adenylyation: Attack on -P
Alanine
NH 3
H3C
C
O
O
C
O
O
H
P
O
O
O
P
O
O
O
P
O
Ribose
Adenine
O
O
NH 3
H3C
C
C
H
O
O
O
P
O
O
O
Ribose
Adenine
+
P
O
O
O
P
O
O
Adenylation: Attack on -P
O
NH 3
H3C
C
C
H
O
O
O
P
O
O
Ribose
Adenine
+
P
O
O
O
P
O
O
Aminoacyl adenylate
–Fatty acid activation
for oxidation
-Amino acid activation
for protein synthesis
Pyrophosphate
PPi
2Pi
O
Pyrophosphorylation: Attack on -P
O
O
P
CH2
O
O
O
O
OH
O
P
O
O
P
O
O
P
O
O
O
OH OH
Ribose-5-phosphate
O
O
P
AMP
CH2
O
O
O
O
P
OH OH
O
O
O
P
O
O
5'-phosphoribose-1-pyrophosphate
(PRPP)
Ribose
Adenine
Phosphorylation: Attack on -P
O
H2C
O
O
OH
O
P
O
P
O
P
O
O
OH
HO
O
O
OH
OH
Glucose
ADP
O
H2C
O
P
O
O
O
OH
HO
OH
OH
Glucose-6-phosphate
O
Ribose
Adenine
Amino Acid Sidechains as
Nucleophiles
O
O
P
O
CH2
O
O
O
N
NH
N
P
O
O
P-lys
(-amino)
O
P
P
O
P-ser, thr
O
O
O
P-his
(1-N)
O
NH C
NH 2
P-arginine
NH
O
P
O
NH C
NH
NH 2
P-creatine
CH2 COO
Enzymatic Phosphoryl Transfers
• Four classes
– Phosphatases
• Water is acceptor/nucleophile
– Phosphodiesterases
• Water is acceptor/nucleophile
– Kinases
• Nucleophile is not water
– Phosphorylases
• Phosphate is nucleophile
Phosphatases: Glucose-6Phosphatase
CH 2OH
O
Enz
X
O
P
O
O
HO
O
CH 2
OH
OH
O
Enz
O
OH
HO
OH
OH
OH
X
P
O
O
Covalent E-S intermediate is formed
X=His
Phosphatases: Glucose-6Phosphate
O
Enz
X
P
O
O
H
H
O
Enz
X
O
HO
P
O
O
Phosphodiesterases: RNAase
O
Pyr
O
H
O
H
H
O
H
OH
P
O
O
H
H
O
H
P
O
Base
O
O
H
O
O
H
Pyr
H
H
O
H
OH
P
O
O
O
O
2',3'-cyclic phosphate
No covalent intermediate
with enzyme
Phosphodiesterases: RNAase
O
Pyr
O
H
H
O
P
O
H
H
O
Pyr
O
H
O
O
O
H
H
O
H
H
O
H
OH
P
O
OH
Kinases: -Phosphoryl
Transfer
• Transfer from ATP
O
O
RX
P
O
O
O
P
O
O
O
P
O
Ribose
Adenine
O
Mg 2+
O
O
RX
P
O
O
+ O
P
O
O
O
P
O
Mg 2+
O
Ribose
Adenine
Kinases: P-Enzyme
Intermediates
O
Enz
X
O
P
O
O
O
P
O
O
O
P
O
ADP
O
Enz
X
P
O
O
O
Ribose
Adenine
Kinases: P-Enzyme Intermediates
O
Enz
X
P
O
Nulceophilic
substrate
Y
O
O
Enz
X
O
P
O
Product
Y
Kinases
RX
R-OH
Example
Hexokinase
PFK
R-OP
Nucleoside
disphosphokinase
R-NH2
Creatine kinase
R-COO–
Succinate
thiokinase
O
Pyruvate kinase
R C COO
Protein-ser-OH Protein kinase
Protein-thr-OH
Enz-X-P?
?
No
Yes
No
Yes
No
Yes
Pyruvate Kinase
• Makes ATP (∆Gº= –31 kJ/mol) from PEP
O
O
H2C
O
C
P
O
O
P
O
COO
O
P
O
O
O
O
O
H3C
O
C
O
Pyruvate COO
P O
O
ADP
+
H2C
C
COO
PEP
∆Gº= –62 kJ/mol
Ribose
Adenine
Phosphoryl-Group Transfer
Potential
Compound
² Gº
PEP
-62 kJ/mole
1,3-bisphospho-49
glycerate
P-creatine
-43
Acetyl-P
-42
ATP (and other
-31
NTP)
Glucose-1-P
-21
Glucose-6-P
-14
Glycerol-1-P
-9
Structure
Enol-P
Acyl-P
Guanidinium-P
Acyl-P
P-anhydride
Hemiacteal-P
Alcohol-P
Alcohol
Significance of “High-Energy” P
Compounds
• Drive synthesis of compounds below
• Phosphated compounds are more
reactive
– Thermodynamically
– Kinetically
• If organism has ATP (etc…), it can do
work and resist entropy
Cells must get ATP