This document contains the syllabus and lecture schedule for a course titled "Science of Living Systems". The course covers topics related to protein structure and function including nucleic acids, transcription and translation, enzymes, photosynthesis, respiration, cellular architecture, and cell division. Lectures are scheduled twice a week and include topics like nucleic acids, transcription and translation, protein structure, enzymes, photosynthesis, respiration, and more. The document also lists required textbooks and provides an overview of protein classification, structure, and enzymatic kinetics including Michaelis-Menten kinetics.

1. Science of
Living System
Soumya De
School of Bio Science
Email: somde@iitkgp.ac.in
Tel: 03222-260514
BS20001

2. Lecture Date Topic
1 20/7/16 Nucleic acids
2 27/7/16 Transcription and Translation
3 3/8/16 Protein structure
4 10/8/16 Enzymes
5 17/8/16 Photosynthesis
6 24/8/16 Respiration
** 31/8/16 CLASS TEST-1
** 7/9/16 DISCUSSION AND REVISION
** 14/9 to 21/9 MID-SEM EXAM
7 28/9/16 Cellular architecture
8 5/10/16 Cell division and apoptosis
** 12/10/16 Autumn Break
9 19/10/16 Host defense/Disease biology/vaccines/antibiotics
10 26/10/16 Responses of living systems/scaling factors
12 2/11/16 Recombinant DNA Technology & its impact
** 9/11/16 CLASS TEST-2
** 16/11/16 DISCUSSION AND REVISION

3. Books Followed:
• How Proteins Work (Mike Williamson)
• Introduction to protein structure (Carl
Branden & John Tooze)
• Biochemistry (Lubert Stryer)
Protein Structure, Function, Kinetics
and Energetics

4. Hierarchy of Protein Structure

5. Protein classification by function
Enzymes: catalyze chemical reactions.
Regulatory proteins: bind to protein receptors, e.g. hormones such as
insulin.
Transport proteins: e.g. myoglobin and hemoglobin transport O2.

6. Hemoglobin – O2 carrier

7. Protein classification by function
Enzymes: catalyze chemical reactions.
Regulatory proteins: bind to protein receptors, e.g. hormones such as
insulin.
Transport proteins: e.g. myoglobin and hemoglobin transport O2.
Storage proteins: e.g. casein in milk, ovalbumin in eggs.
Contractile and motile proteins: involved in motion, e.g. myosin and actin in
muscle.

8. The major proteins of muscle are
myosin and actin

9. Protein classification by function
Enzymes: catalyze chemical reactions.
Regulatory proteins: bind to protein receptors, e.g. hormones such as
insulin.
Transport proteins: e.g. myoglobin and hemoglobin transport O2.
Storage proteins: e.g. casein in milk, ovalbumin in eggs.
Contractile and motile proteins: involved in motion, e.g. myosin and actin in
muscle.
Structural proteins: e.g collagen, keratins (in skin, hair), elastin (vocal
chord, arteries), silk.
Binding/Interaction proteins: proteins bind one another only when a signal
is received, e.g. phosphorylation of Insulin Receptor Substrate (IRS) protein.
Protective proteins: e.g. immunoglobulins, proteins of blood clotting system.

10. Antibodies recognize antigens

11. Enzymes – Biological catalysts
• First discovered by Eduard Buchner in 1897 who
observed that yeast extracts can ferment sugar to
alcohol. Nobel Prize 1907.
• This proved that fermentation was promoted by
molecules that continued to function when removed
from cells.
• The first enzyme to be purified and crystallized was
urease in 1926 by James Sumner at Cornell University;
these crystals consisted entirely of protein. Nobel Prize
1946.
• Later, pepsin, trypsin and other digestive proteins were
isolated and determined to be purely protein as well.

12.  Enzymes are the catalysts of nature.
 With the exception of catalytic RNA, all enzymes are
proteins.
 Catalyst alter the rate of a chemical reaction without
undergoing a permanent change in structure.
 Catalytic activity is dependent upon native (i.e. folded)
conformation of the enzyme; if it is lost, then catalytic
activity is lost as well.
 All levels of protein architecture (i.e. primary to
quaternary structure) must be intact and correct for
enzymes to perform their functions.
 They range in molecular weights from 12,000 to over 1
million daltons.
Enzymes

13. Enzymes
Most enzymes are proteins.
Simple Enzymes: composed of whole proteins.
Complex Enzymes: composed of protein plus a relatively
small organic molecule.
holoenzyme = apoenzyme + prosthetic group or coenzyme

14. Enzymes
Most enzymes are proteins.
Simple Enzymes: composed of whole proteins.
Complex Enzymes: composed of protein plus a relatively
small organic molecule.
holoenzyme = apoenzyme + prosthetic group or coenzyme
A prosthetic group describes a small organic or metalloorganic
molecule bound to the apoenzyme by covalent bonds.
When the binding between the apoenzyme and non-protein
components is non-covalent, the small organic molecule is
called a coenzyme.
Coenzymes serve as transient carriers of specific functional
groups.
They often come from vitamins (organic nutrients required in
small amounts in the diet)

15. Oxidoreductases Add or remove hydrogen atoms (transfer of electrons).
Transferases
Transfer functional groups between donor and
acceptor molecules. E.g. Kinases transfer phosphate
group from ATP to other molecules.
Hydrolases
Catalyze hydrolysis i.e. breaking of a bond by the
addition of a water molecule.
Lyases
Add water, ammonia or carbon dioxide across double
bonds, or remove these elements to produce double
bonds.
Isomerases
Carry out many kinds of isomerization. E.g. L to D
isomerizations, mutase reactions (shifts of chemical
groups) and cis-trans isomerization of peptide bonds.
Ligases
Catalyze reactions in which two chemical groups are
joined (or ligated) with the use of energy from ATP.
International Classification of Enzymes

16. How enzymes work
• Enzymes catalyze chemical reactions that do not normally proceed under conditions
such as neutral pH, mild temperature, and aqueous solvent.
• The site of catalytic activity on the enzyme is known as the active site.
• The molecule that binds to the active site and is acted upon by the enzyme is called
the substrate.
• The two together form what is known as the enzyme-substrate complex
• The function of an enzyme is to increase the rate of a chemical reaction without
affecting its equilibrium.
• Therefore, enzymes don’t make more product, they just make product faster.
enzyme-substrate complex

17. Active Site
• The area of an enzyme where the substrate
binds.
• Structure has a unique geometric shape that
is designed to fit the molecular shape of the
substrate.
• Active sites contain residues that bind the
substrate and also participate in catalysis.
• Active sites sometimes contain a co-factor.
• Active site residues have several important
properties:
– Charge (partial, dipoles, helix dipole)
– pKa
– Hydrophobicity
– Flexibility
Chymotrypsin

18. Substrate Binding site (Active site)
Complementarity
• Geometric
• Electronic (electrostatic)
• Stereospecificity (enzymes
and substrates are chiral)
1. Lock and Key model
2. Induced Fit model

19. • An enzyme binds a substrate in a region called the active site.
• The active site shape is complementary to the substrate i.e. not all
substrates can fit the active site.
• Amino acid sidechains in the active site bind the substrate.
Lock and Key Model
• Enzyme structure is flexible, not rigid.
• Enzyme and active site adjust their shape to bind the substrate.
• Increases range of substrate specificity.
• Shape changes also improve catalysis during reaction
- by stabilizing the transition-state.
Induced Fit Model
Substrate Binding site (Active site)

20. Enzyme-Substrate Interaction

21. Factors that influence enzyme activity -
temperature

22. Factors that influence enzyme activity - pH

23. ΔGǂ
S→Pfor uncatalyzed reaction = 107 kJ
ΔGǂ
cat for catalyzed reaction = 47 kJ
kuncat  e-107000/8.314x298
kcat  e-47000/8.314x298
kcat/kuncat = ~5x1010
How enzymes work?
k  e-ΔG /RTǂ
1 sec ~1500 years

25. How can an enzyme reduce the
activation energy?
(1) Binding to the substrate can be done such that the
formation of the transition state is favored
(2) Orientation and positioning of substrate(s)
(3) Bonds in the substrate can be ‘activated’ by functional
groups in the catalytic site

26. How can an enzyme reduce the
activation energy?

27. - the activation energy for the formation of the intermediate state, and its
conversion to the final product are each lower than the activation energy for the
uncatalyzed reaction
-intermediate state- resembles transition state but with lower energy, (due to
interaction with a catalyst)
- transition state defines free energy maximum state
Binding energy helps reduce
the activation energy

28. How enzymes work? Example: Chymotrypsin
• Chymotrypsin is a digestive enzyme component of pancreatic juice.
• It performs proteolysis i.e. the breakdown of proteins and polypeptides.
• Chymotrypsin preferentially cleaves peptide amide bonds where the
carboxyl side of the amide bond is an aromatic amino acid (tyrosine,
tryptophan, and phenylalanine).
• Chymotrypsin facilitates the nucleophilic (OH-) attack from the correct
orientation.
• It lowers the transition state energy.
• Chymotrypsin favors reaction ‘b’ after formation of the intermediate.
N N NN
N
N

29. How enzymes work? Example: Chymotrypsin

30. How enzymes work? Example: Chymotrypsin

31. How enzymes work? Example: Chymotrypsin

32. E = Enzyme S = Substrate P = Product
ES = Enzyme-Substrate complex
k1 rate constant for the forward reaction
k-1 = rate constant for the breakdown of the ES to
substrate
k2 = rate constant for the formation of the products
Enzyme Kinetics
E  S
k1
k1
   ES
k2
  E  P

33.    ES
P
2k
dt
d
v 
        ESESSE
ES
211 kkk
dt
d
 
1
E  S
k1
k1
   ES
k2
  E  P
Rate of formation of product P:
Rate of formation of enzyme-substrate complex ES:

34.   
 ES
SE
1
1
 
k
k
KD
KD is the dissociation constant for the ES complex.
Formation of enzyme-substrate complex
E  S
k1
k1
   ES
k2
  E  P
At equilibrium:
forwards reaction rate = reverse reaction rate
i.e. k1[E][S] = k-1[ES]

35. Assumption of steady-state
Transient phase where in the course of a reaction the
concentration of ES does not change
  0
ES

dt
d
2
E  S
k1
k1
   ES
k2
  E  P

36.      ESEE T  3
Combining 1 + 2 + 3
        ESkkSES-Ek 21-T1 
        SEkSkkkES T1121- 
rearranging
Divide by k1 and solve for [ES] Where
1
21-
k
kk
K

M     
 SK
SE
ES T


M

37.        
 SK
SE
ES
P T2
2
0 







 Mt
o
k
k
dt
d
v
vo is the initial velocity when the reaction is just starting out.
And is the maximum velocity T2max EkV 
 
 SK
SVmax


M
ov
The Michaelis - Menten
equation

38. Michaelis – Menten Kinetics
The Km is the substrate concentration
where vo equals one-half Vmax

39. low [S], v is proportional to [S] - first order
high [S], v is independent of [S] - zero order
Michaelis – Menten Kinetics

40. The KM widely varies among different enzymes
The KM
can be expressed as: 1
2
1
2
1
1
KK
k
k
k
k
k
k
DM  
As KD decreases, the affinity for the substrate
increases. The KM can be a measure for substrate
affinity if k2<<k-1
The parameters vmax and KM are used to
compare enzyme activities

41. V0 = Vmax [S]
Km + [S]
Lineweaver-Burk plot transforms the Michaelis-
Menten equation into linear form.
1 = Km + [S]
V0 Vmax [S]Lineweaver-Burk Plot
The double reciprocal plot
Km 1 1
Vmax [S] Vmax
= +
1
V0
(y = mx + c)

42. For small errors in at low [S]
leads to large errors in 1/vo

43. For Michaelis - Menton kinetics k2= kcat
When [S] << KM very little ES is formed and [E] = [E]T
and
      SE
K
k
SE
K
k
M
cat
T
M
2
ov
kcat/KM is a measure of catalytic efficiency
V0 = Vmax [S]
KM + [S]   kcatk 2
T
max
E
V

The turnover number (kcat)
kcat is how many reactions an enzyme can catalyze per second

44. KM
Relates to how strongly an enzyme binds its substrate.
High KM means strength of binding is low.
kcat
Relates to how rapid a catalyst the enzyme is.
High kcat means high speed of catalysis.
Vmax
Related to kcat and [ET] by: Vmax=kcat[ET]
High Vmax means high rate of catalysis.

45. A high kcat/KM ratio implies an efficient enzyme
This could result from: Large kcat
Small KM
• kcat = turnover number; kcat = Vmax/[ET]
• kcat/Km is a measure of activity, catalytic efficiency
KM is a useful indicator of the affinity of an enzyme
for the substrate
A low KM indicates a high affinity for the substrate

46. Enzyme Inhibition
• Inhibitors: compounds that decrease or eliminate activity of an
enzyme.
• Can decrease binding of substrate (affect KM), or turnover number
(affect kcat) or both.
• Most drugs are enzyme inhibitors.
• Inhibitors are also important for determining enzyme mechanisms
and the nature of the active site.
Some examples of enzyme inhibitors:
• Antibiotics inhibit enzymes by affecting bacterial metabolism.
• Nerve Gases cause irreversible enzyme inhibition.
• Insecticides – choline esterase inhibitors.
• Many heavy metal poisons work by irreversibly inhibiting enzymes,
especially cysteine residues.

47. Types of Enzyme Inhibition
• Reversible inhibition
reversibly bind and dissociate from enzyme,
activity of enzyme recovered on removal of
inhibitor - usually non-covalent in nature
– Competitive
– Uncompetitive
– Noncompetitive (Mixed)
• Irreversible inhibition
irreversibly associate with enzyme. Activity of
enzyme not recovered on removal - usually
covalent in nature.

48. Competitive Inhibition
• Inhibitor competes for the substrate binding site
• most look like substrate
• substrate mimic / substrate analogue

49. Competitive Inhibition
(y = mx + c)
Km 1 1
Vmax [S] Vmax
= +
1
V0

50. Competitive Inhibition
(y = mx + c)
Km 1 1
Vmax [S] Vmax
= +
1
V0
α = 1 + [I]/Ki

51. Competitive Inhibition
No Reaction
• Methanol poisoning is treated with ethanol; the formation of
formaldehyde is slowed and spread out over a longer period of time,
lessening its effects on the body

52. Uncompetitive Inhibition
Uncompetitive inhibitors bind at a site distinct from the substrate active site
and bind only to the ES complex
• Active site distorted after binding of S (usually
occurs in multisubstrate enzymes) Decreases both
KM and kcat
• Vo = Vmax[S]/(KM + ’[S]) K’I = [ES][I]/[ESI]
• Cannot be reversed by increasing [S] – available
enzyme decreases

53. Uncompetitive Inhibition
(y = mx + c)
α’ = 1 + [I]/K’i
Km 1 1
Vmax [S] Vmax
= +
1
V0

54. Inhibitor can bind at a site distinct from the substrate active site
to either E or ES.
Mixed (Non-competitive) Inhibition
• Vo
= Vmax[S]/(KM + ’[S])
• Vmax decreases; KM can
go up or down.

55. Non-competitive Inhibition
(y = mx + c)
Km 1 1
Vmax [S] Vmax
= +
1
V0

56. Mixed inhibition refers to a combination of two different types of reversible enzyme
inhibition – competitive inhibition and uncompetitive inhibition. The term 'mixed' is used
when the inhibitor can bind to either the free enzyme or the enzyme-substrate complex.
In mixed inhibition, the inhibitor binds to a site different from the active site where the
substrate binds. Mixed inhibition results in a decrease in the apparent affinity of the
enzyme for the substrate (Km
app > Km, a decrease in apparent affinity means the Km
value appears to increase) and a decrease in the apparent maximum enzyme reaction
rate (Vmax
app < Vmax).
Mathematically, mixed inhibition occurs when the factors α and α’ (introduced into the
Michaelis-Menten equation to account for competitive and uncompetitive inhibition,
respectively) are both greater than 1.
In the special case where α = α’, noncompetitive inhibition occurs, in which case
Vmax
app is reduced but Km is unaffected. This is very unusual in practice
Non-competitive inhibition models a system where the inhibitor and the substrate
may both be bound to the enzyme at any given time. When both the substrate and the
inhibitor are bound, the enzyme-substrate-inhibitor complex cannot form product and
can only be converted back to the enzyme-substrate complex or the enzyme-inhibitor
complex. Non-competitive inhibition is distinguished from general mixed inhibition in
that the inhibitor has an equal affinity for the enzyme and the enzyme-substrate
complex.

57. Lineweaver-Burke plots - Summary

58. Allosteric regulation of enzymes
A small molecule can bind an enzyme and act as an effector
or regulator to activate or inactivate its function.
In such case, the protein is said to be under allosteric control.
If the binding of the small molecule (ligand) is distant from the
protein’s active site and regulation is a result of a
conformational change in the protein when the ligand is
bound, the regulation is called allosteric regulation.
Many types of proteins show allosteric control:
- hemoglobin (NOT myoglobin)
- various gene-regulating proteins (transcription factors)
e.g. Lac repressor (see lecture 2)

59. Phosphofructokinase (PFK-1) and ATP
Substrate: Fructose-6-phosphate
fructose-6-phosphate + ATP  fructose-1,6-bisphosphate + ADP
phosphofructokinase-1

60. Allosteric regulation of PFK-1
• PFK-1 catalysed reaction lies near the beginning
of the respiration pathway in cells.
• The end product of respiration is synthesis of
ATP, the energy currency.
• If there is a lot of ATP in the cell PFK-1 is
inhibited. Then respiration slows down and less
ATP is produced.
• As ATP is used up PFK-1 inhibition stops and
the reaction speeds up again.

61. The switch: Allosteric inhibition
Allosteric means “other site”
E
Active site
Allosteric
site

62. Allosteric inhibition: Switching off the enzyme
• These enzymes
have two receptor
sites
• One site fits the
substrate like other
enzymes
• The other site fits an
inhibitor molecule Inhibitor fits
into allosteric
site
Substrate
cannot fit
into the
active site
Inhibitor
molecule

63. E
Active
site
Allosteric
site empty
Substrate fits into the active site
The inhibitor
molecule is
absent
Conformational
change
Inhibitor fits into
allosteric site
Substrate
cannot fit
into the
active
site
Inhibitor molecule
is present
E
Allosteric inhibition: Switching off the enzyme

64. Conformational change: Change in shape of
the protein
• When the inhibitor is present it fits into its site
and there is a conformational change in the
enzyme molecule
• The enzyme’s molecular shape changes
• The active site of the substrate changes
• The substrate cannot bind with the substrate

65. • Irreversible inhibitors are those that combine with or
destroy a functional group on an enzyme that is
essential for activity
• They usually form covalent linkages to the enzyme
Diisopropylfluorophosphate binds irreversibly with
chymotrypsin at the Ser195 residue; this gives info
justifying this as the primary active site of the enzyme
Irreversible inhibition: Kill the enzyme

66. • A special class of irreversible inhibitors is the suicide
inactivators
• These are unreactive until bound to the active site
• They are designed to carry out the first few steps of a
normal enzyme reaction, but instead of forming a
product, they form a highly reactive compound that
binds irreversibly to the enzyme
• They are sometimes called mechanism-based
inactivators, because they use the normal enzyme
mechanism to lead to the inactivation
• These are often used in drug design