BCH2333 – Introduction to Biochemistry

Lecture 4

Thermodynamics

Nucleotides

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Quantitation of pI:
The pI can be quantified by the Henderson-Hasselbalch equation:
-1 +1
The average of the pKas relating to the
ionization of the zwitterion

Glycine Net Charge

Aspartic Acid Net Charge

-1

-2
0

-1
0
+1
+1

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pI of Proteins (polyampholytes)
Proteins contain up to hundreds of pH-responsive groups; when the net charge of the
protein is 0, anions balance cations and the pI is reached

Three conclusions to be applied to proteins and pI:

@ pH<pI, |S(q-)|<|S(q+)|, @ pH=pI, |S(q-)|=|S(q+)|, @ pH>pI, |S(q-)|>|S(q+)|,

net charge=+ve net charge=0 net charge=-ve

=anionic =cationic
This has a major impact on protein solubility and
biomolecular interactions: why?
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pI of Proteins (polyampholytes)
This has a major impact on protein solubility and biomolecular interactions: why?

Non-covalent intermolecular interactions

dictate the function of even large
biomolecules.
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pH Extra Resources:

https://www.khanacademy.org/science/chemistry/oxidation-reduction/acids-and-bases/v/pka-and-pkb-
relationship

http://yeahchemistry.com/tutorials/Acid-Base

http://mysite.science.uottawa.ca/aflynn/Organic_Acid-Base.html

https://www.mcb.ucdavis.edu/courses/bis102/acid-base/

Buffers Extra Resources:

http://chemcollective.org/activities/tutorials/buffers/buffers1

https://www.khanacademy.org/science/chemistry/oxidation-reduction/acids-and-bases/v/buffers-and-
hendersen-hasselbalch

https://www.youtube.com/watch?v=MlH-qE-Xki8

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Free Energy:
DG=DH-TDS

Gibbs Free Energy Enthalpy Entropy

The overall energy The absorption (>0) The degree of
change of a process, or release (<0) of randomness of a
and whether it energy during a system (disorder)
requires energy or reaction, measured in
gives off energy the form of heat
(measure of useful evolution
work)
DG, DH, and DS are all state functions: they depend on the initial and final states of
the system under study and not on the pathway taken between states.
In bioenergetics, thermodynamics (2nd law) allows us to tell two things about a
process:
1. Is it reversible?
2. Is it favored to
occur?
Importantly: Biological systems are open (i.e. they exchange energy with their
surroundings), and operate at constant(ish) temperature and pressure.
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How we (BCH2333A) use thermodynamics:

B C

A D

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Free Energy Derivation:

Because biology operates in an open system, the entropy of the system and the
surroundings must be considered:

We are only interested in the system under study. We know that the system affects
the surroundings through heat exchange (constant P, @ temperature T):

DG is the measure of the disorder of the

universe at a given temperature T
We know that DSuniverse>0, therefore:

(a) Processes that result in DSuniverse>0 will

yield a DG<0
(b) Processes that decrease the energy of
a system (DH<0) favour DG<0
(c) Processes that increase the disorder
of a system (DS>0) favour DG<0
(d) Processes where DH=TDS result in
DG=0 and a reversible system at
equilibrium
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Thermodynamic Outcomes for Biochemistry:

Two important points to remember about free energy:

1. The thermodynamic favorability of a reaction says nothing about the rate of the
reaction.
The rate can be increased in the favored direction by a catalyst

2. In biochemistry entropy can and does decrease in favorable reactions

initial initial

final
initial

final final

DS can decrease as long as energy is expended (DH<0) to pay the price for
organization.
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DG, Equilibrium, & Concentration:

The magnitude of DG is a measure of how far a process is from equilibrium; In a cell,
the processes are not in equilibrium (Why not? Are they ever?)

DG is dependent on the equilibrium position of a given system:

aA + bB @ equilibirum
cC + dD
DG

aA + bB Not equilibirum

cC + dD
DG°, standard free energy change; @ P=1 bar, T=293 K, [solute]=1M
Used as a point of reference for real non-equilibrium
systems
By Le Chatelier’s Principle, the equilibrium will reestablish due to the increase in
reactant A, and the magnitude of this driving force towards equilibrium is described
by DG
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DG, Equilibrium, & Concentration:

Since

R = gas constant

We know that DG is a state function, where

DG=Gfinal-Ginitial
Standard Actual
aA + bB reference value concentrations
cC + dD for free energy of the reactants

If the system is at equilibrium, we know

that (a) DG=0 and (b) Q=K:

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DG, Equilibrium, & Concentration:

Normal, “healthy” conditions in biochemistry are within a normal range of
temperature, pH, and solute concentrations known as homeostasis.
Homeostasis is not Equilibrium

Homeostasis is favourable, and

requires energy to maintain
(where does this energy come
from?)

Allows for a quantitative

prediction of whether a reaction
will proceed, and in what direction
it will proceed

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DG, Equilibrium, & Concentration:

Aldolase catalyzes the following reaction:
F-1,6-BP  G-3-P + DHAP ΔGo’=23.8 kJ/mol
If [F-1,6-BP] = 1.4 X 10-5 M, [G-3-P] = 3 μM, and [DHAP] = 1.6 X 10-5 M, at body temperature,
what is the actual free energy change for the reaction? R=8.315 X 10 -3 kJ/molK

[ 𝐺 3 𝑃 ] [ 𝐷𝐻𝐴𝑃 ] ( 3 ×10− 6 𝑀 )(1.6 × 10−5 𝑀 )

𝑄= = =3.4 ×10− 6
[ 𝐹 16 𝐵𝑃 ] 1.4 ×10 −5 𝑀

∆ 𝐺=23.8
𝑘𝐽
𝑚𝑜𝑙 (
+ 8.315 ×10 −3
𝑘𝐽
𝑚𝑜𝑙 ∙ 𝐾 )
( 310 𝐾 ) ln ( 3.4 ×10− 6 )

𝑘𝐽
∆ 𝐺=− 8.6
𝑚𝑜𝑙

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Driving Biochemical Reactions:

Unfavorable reactions exist throughout biochemistry, but are driven in their

forward direction because :
1. Cells maintain Q/K<1 by consuming products as fast as they are formed,
and/or keeping reactant levels very high

A
C
B versus A B
C
2. Coupling unfavorable reactions to favorable reactions
Unfavorable but
A DG ’>0
o
necessary for life

+ B
C DGo’<<0 E.g. Organic
phosphate hydrolysis
D
A+C DGo’<0
B+D
**DGo’= biochemical standard state where [H +]=10-7 M

High energy phosphate bonds are common sources of chemical energy

in biochemistry. Its one form of how we store our energy taken in by
nutrition, and it is how food energy is used to power life.
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Driving Biochemical Transport:

The same principles used to drive unfavorable reactions exist to drive the
transport of solutes across biological membranes.

DG Outcome
[A]1>[A]2 <0 Favorable
[A]1<[A]2 >0 Unfavorable
[A]1=[A]2 =0 Equilibrium

As with reactions, transport is

DG=Gfinal-Ginitial
driven by:
1. Maintaining [A]1>[A]2
2. Coupling unfavorbale transport
conditions to favorable transport
or reactions (ATP-driven)

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Primary Structure of Nucleic Acids:

Nucleic acids are composed of 3
parts:
1. Sugar – scaffold
2. Phosphate – linker
5’ 3. Base - code

Sugar:
5-carbon, b isomer at anomeric C

3’ b-D-ribose b-D-2-deoxyribose
(RNA) PO43- (DNA)

CH OH CH2OH Base
5’ 2
OH
O O OH
4’ H H 1’ H H
H H H H
3’ 2’
HO OH HO H

Phosphodiester link forms between 5’ OH of one unit PO43-

and 3’ OH of preceding unit. Phosphate has a pKa~1,
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Primary Structure of Nucleic Acids:

Phosphodiester-linked sugars are the structural components of DNA comprising the
backbone does not encode information.

Genetic information is encoded in the heterocyclic nucleobase

Position of
glycosidic
bond to
anomeric C-
1’

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Primary Structure of Nucleic Acids:

Base + Sugar Base + Sugar + Phosphate

Nucleic acids can exist as
higher order phosphate
conjugates (i.e.
diphosphate and
triphosphate).

While these higher order

structures are not present
themselves in
polynucleotides, they are
important drivers for the
linkage of nucleotides
(more to come soon…).

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Conformations of Nucleic Acids:

There are three important chemical considerations defining the structure and function
of polynucleotides:

1. The sugar conformation can change as the 5-membered pentose ring is flexible
and prone to twisting (or puckering).
The asymmetry of the pentose ring results in two preferred conformations,
which can differentiate DNA from RNA
Glycosidic Glycosidic
Bond Bond

Endo face P C5’ Endo face

(above) C2’ base (above)
O4’ base C5’ C3’ O4’
C4’ P
C1’ C1’
C4’
7.0Å 5.9Å P
C2’-endo C3’-endo
P

DNA RNA
C2’-endo
base
base OH C3’-endo base
base

OH

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Conformations of Nucleic Acids:

There are three important chemical considerations defining the structure and function
of polynucleotides:

1. The sugar conformation can change as the 5-membered pentose ring is flexible
and prone to twisting (or puckering).
2. Steric constraints hinder rotation around the glycosidic bond, favoring the anti
conformation (with exceptions…)
Purines Pyrimidines

Steric Clash
(O2-O4’)

P P P P

P P P P
Anti Syn Anti Syn

The anti conformation is favored in the double helix due to interstrand bonding. For guanosine, there
is a favorable interaction between the N2 and 5’-PO 42- that stabilizes the syn conformation. This can
result in unusual DNA helices that we will see shortly.
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Conformations of Nucleic Acids:

There are three important chemical considerations defining the structure and function
of polynucleotides:
1. The sugar conformation can change as the 5-membered pentose ring is flexible
and prone to twisting (or puckering).
2. Steric constraints hinder rotation around the glycosidic bond, favoring the anti
conformation (with exceptions…)
3. Tautomerization can occur with the keto and amine conformations favored, which
directly impact H-bonding

Keto Enol Amine Imine

(favored) (favored)

Tautomer: isomers that differ in the location of protons (and consequently double bonds)
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Naming of Nucleic Acids:

Base
Nucleoside
Adenine Adenosine
Guanine Guanosine
Cytosine Cytidine
Thymine Thymidine
Uracil Uridine
A= adenosine dA = deoxyadenosine
but T = deoxythymidine (DNA only)
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Formation of Polynucleotides:
Polynucleotide chains do not form through dehydration,
since this reaction is thermodynamically unfavored.

The metastability of DNA and RNA allow for controlled

-H2O turnover and reorganization via base catalysis (e.g.
DG =25 kJ/mol nucleases). More to come on this…
o’

+H2O
cat. -OH
2 DGo’PPio-PO4=-19 kJ/mol

Metastable

How do polynucleotides form? Coupled reactions!

Breaking high energy phosphate bonds (NTP
to NMP+PPi, and PPi to orthophosphate)
provides a favorable thermodynamic driving
force to yield polynucleotides DGo’NTPNMP=-31 kJ/mol

NTP + H2O NMP + PPi DGo’=-31 kJ/mol

DNAN+NMP DNA(N+1)+H2O DGo’=25 kJ/mol
PPi + H2O 2 HPO42- DGo’=-19 kJ/mol
DGo’Polynucleotide=25 kJ/mol
DNAN+NTPDNA(N+1)+2HPO42- DGo’=-25 kJ/mol
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Primary Structure of Nucleic Acids: Sequence =

NH2 Genetic
O
- Information
Can differentiate DNA from RNA by
-O P O
N
looking for 2’-OH and bases in sequence
(U vs. T) O
O
N O C

Convention for writing is from 5’ to 3’ NH2

O N
Purines connected to sugar phosphate
-O P O
N

backbone at N9 O N N
O A

Pyrimidines connected to backbone by N1 O

O N
-O P O
NH
All bases are connected to anomeric C
(C1’) by b-linkage O N N NH2 G
O

NH2

C A G C O N
3’
-O P O
5’ End 3’ End
N O
C
O
O

5’
5’-CAGC-3’ OH
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Primary Structure of Nucleic Acids:

In biochemistry, there are structural layers of complexity to provide flexibility to
biomolecular function and control of function. This is critical for proteins (to come)
and for nucleic acids.
For nucleic acids, the primary structure has both directionality (5’3’) and
individuality (sequence of bases)

We talked before about chemical evolution and the maintenance of biomolecule

complexity through complementarity. This is key for nucleic acids!

Intramolecular 5’ 3’ Sense

Complimentarity

3’ 5’ Anti-sense

“Monomer Sea”
+ Two characteristics about DNA facilitate this
chemical evolution and fidelity of replication:
1. Two anti-parallel strands (sense and
antisense)
Intermolecular 2. Specific base pairing
Complimentarity
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Primary Structure and Base Pairing:

5’ 3’ Sense Two characteristics about DNA facilitate this
chemical evolution and fidelity of replication:
1. Two anti-parallel strands (sense and antisense)
3’ 5’ Anti-sense 2. Specific base pairing

2 H-bonds
O

3 H-bonds
O

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