.

1. M. Sc. (Organic)
Course: 525F
Bioorganic & Food Chemistry

2. Chapter 1
Enzyme Catalyzed Reaction
Enzyme: Enzymes are a group of proteins which exhibit catalytic property
in biological reactions providing driving force to the reaction.
Example: Urease catalyzes hydrolysis of urea to ammonia and CO2
Lipases act on lipid
Lactase acts on lactose
Proteases act on proteins

3. Co-factors: Cofactors are non protein compounds bound to the enzymes and
required for enzyme activity. Organic molecule or different metal ions are acted
as cofactors of enzymes. The cofactors are generally stable to heat, whereas most
enzyme proteins loss activity on heating.
Example:
Zn2+ for alcohol dehydrogenase, carbonic anhydrase, carboxy peptidase
Mn2+
Mg2+
Fe2+ or Fe3+
Cu2+
K+
Na+

4. Role of metal ion cofactors:
(1) To serve as primary catalytic center
(2) To act as a bridging group, to bind substrate and enzyme tighter through
formation of a coordination complex
(3) As an agent to stabilize the confirmation of enzyme protein in its catalytic
active form.
Metalloenzymes: The enzymes require metal ions are sometimes called
metalloenzymes.

5. Co-enzymes: The organic cofactors bound tightly by covalent linkage to the
enzyme are regarded as coenzymes. Some enzymes need both of them
to perform proper catalytic activity.
Prosthetic group: The functional group or site of coenzymes (organic molecule)
which makes covalent linkage to bound tightly to enzyme protein is known as a
prosthetic group.
Role of co-enzymes: Coenzymes usually function as intermediate carriers of
functional groups, of specific atom or electrons that are transferred in the
overall enzymatic reaction.

6. Coenzyme Entity transferred
Nicotinamide adenine
dinucleotide
Hydrogen atoms
(electons)
Biocytin Carbondioxide
Pyrodoxal phosphate Amino groups
Thiamine pyrophosphate aldehyde
Example of coenzymes

7. Holoenzyme: The catalytic active enzyme-cofactor complex is called the
Holoenzyme.
Apoenzyme: The term “apoenzyme” refers to the protein portion of an
enzyme. More specifically, when cofactors are removed from enzyme, the
remaining protein, which is catalytically inactive by itself, is called an
apoenzyme.

8. Classification of enzymes (without code)
1. Oxido-reductases- catalyze oxidation reduction reactions. For example,
A reduced + B oxidized = A oxidized + B reduced
Oxidases: tyrosinase, uricase
Anaerobic dehydrogenases: succinate and lactate dehydrogenases.
Hydroperoxidases: peroxidase, catalase.
2. Transferases- catalyze group transfer reactions by transferring of functional groups. ( one –
carbon group, aldehyde & ketonic groups, acyl groups etc.). Example: hexokinase,
phophopyrolase, amino acid transacetylase.
3. Hydrolases- catalyze hydrolysis reactions on esters, glycoside bonds, peptide bonds, acid
anhydrides. Example, Urease, lipases, phophotases.

9. 4. Lyases- catalyze reactions involving the removal of a group leaving a double bond
or addition of a group to a double bond. example, aldolase, fumerase.
5. Isomerases- catalyze reaction involving isomerization. Racemases, epimerases.
6. Ligases or synthetases- catalyze reaction involving formation of bonds ( C-C, C-S, C-N, C-O)
to link two molecules, coupled with the cleavage of a pyrophosate bond of ATP, or similar
triphosphate. Succinate thiokinase, glutamine synthetase.

10. Look on Back:
(on the basis of number of molecules)
Monomolecular reactions- one molecule undergoes reaction
Bimolecular reactions – two molecules undergo reaction
Trimolecular reactions- Three molecules undergo reaction
Chemical Kinetics
(On the basis of reaction order)
Zero order reaction
First order reactions
Second order reaction- pseudo first order if concentration of one reactant
is very low compared to other one.
Third order reactions
How the reaction rate is influenced by the concentration of reactants?

13. Kinteics of enzyme catalyzed reactions:
The Michaelis - Menten Equation
Note: Enzyme-catalyzed reactions show a distinctive feature not usually
observed in nonenzymatic reactions.
-------------------------------------------------------------------------
Vmax
V0
[Substrate]
----
----------------
½ Vmax
Figure: Effect of substrate
concentration on the rate
of an enzyme-catalyzed
reaction.

14. Hypothesis by A. J. Brown and V. Henri : An enzyme reacts with a substrate
reversibly to form a complex, as an essential step in the catalyzed reaction
General theory of enzyme action and kinetics
(developed by L. Michaelis & M. L. Menten and extended by Briggs and
Haldane): For quantitative analysis of all aspects of enzyme kinetics and
inhibition.
Michaelis and Menten theory assumes that the enzyme E first combines with
the substrate S to form the enzyme-substrate complex ES; the later then
breaks down in a second step to form free enzyme and the product P:
k+1
K-1
ES
E + S
k+2
K-2
E + P
ES
--------------------------- (1)
--------------------------- (2)

15. Derivation of Michaelis-Menten Equation:
We start with the kinetic mechanism shown in the equation 1:
k1
k2
ES
E + S
k3
E + P --------------------------- (1)
In eq 1, S is substrate, ES is the enzyme-substrate complex, and P is product.
Assumptions:
 During the early stages of the reaction so little product is formed that the
reverse reaction (product combining with enzyme and re-forming
substrate) can be ignored (hence the unidirectional arrow under k3).
 The concentration of substrate is much greater than that of total enzyme
([S] >> [Et]), so it can essentially be treated as a constant.

16. The rate of this process (k3[ES]) to the change in product concentration as a
function of time (d[P]/dt), or, equivalently, we can designate the rate with an
italicized v (v) as follows in eq 2:
--------------------------- (2)
The concentration of the enzyme-substrate complex ([ES]) cannot be
measured. Therefore, an alternative expression for this term is needed.
The enzyme that we add to the reaction will either be unbound (E) or bound
(ES) we can express the fraction of bound enzyme as follows:
[ES]
Et
=
[ES]
ES +[E]
d[P]/dt = v= k3[ES]
--------------------------- (3)
[ES]=
Et [ES]
ES +[E]
--------------------------- (4)

17. Now If we assume that enzyme kinetics is the steady-state assumption.
Basically, it says the rate of change of [ES] as a function of time is zero:
d[ES]/dt = 0. Another way to express the steady-state assumption is that the
rate of formation of ES equals the rate of breakdown of ES. We can express
this latter statement mathematically as in eq 6:
k1[E][S] = k2[ES] + k3[ES] = (k2+ k3)[ES]
--------------------------- (5)
--------------------------- (6)
[ES]=
Et
1+ E / ES
--------------------------- (7)
[E]/[ES] = (k2+ k3)/ k1[[S]

18. We now define a new constant, the Michaelis constant (Km), as follows in eq
8:
--------------------------- (8)
--------------------------- (9)
Km=
(𝑘2
+𝑘3
)
𝑘1
--------------------------- (10)
If we substitute Km back into eq 7 we obtain eq 9:
[E]/[ES]=
𝐾𝑚
S
[ES]=
Et
1+𝐾𝑚/ S
We now substitute the ratio Km/[S] from eq 9 in place of the ratio [E]/[ES] in
eq 5 and we obtain eq 10:
Or [ES]=
Et
[S]
𝐾𝑚+ S
--------------------------- (11)

19. we can substitute this alternative expression of [ES] into eq 2 and obtain eq
12:
--------------------------- (12)
--------------------------- (13)
v =
𝑘3
[E𝑡
][S])
𝐾𝑚
+[𝑆]
If we imagine [S] > > Km, then the eq 12 becomes as 13:
The constant k cat of eq 13 is used to signify that k3 is considered the catalytic
constant. Under such conditions, when [S] is said to be saturating, the enzyme
is functioning as fast as it can and we define k3[Et] (or kcat[Et]) to be equal to
Vmax, the maximum velocity that can be obtained. Therefore, eq 12 can be
rewritten into the familiar form of the Michaelis-Menten equation (eq 14):
v =
𝑘3 [E𝑡][S])
[𝑆]
= k3 [Et] = kcat [Et]

20. --------------------------- (14)
--------------------------- (15)
v =
𝑉𝑚𝑎𝑥[S])
𝐾𝑚
+[𝑆]
Again, we imagine what happens when Km > > [S] as follows in eq 15:
Since kˈ = Vmax/ Km in eq 15, we refer to Vmax/ Km as an apparent (or pseudo)
first order rate constant.
Another way to look at a similar, related concept is to rewrite eq 12 as
follows:
v ≈
𝑉𝑚𝑎𝑥[S])
𝐾𝑚
= k ˈ[S]
v =
𝑘𝑐𝑎𝑡
[
E𝑡][S])
𝐾𝑚+ [𝑆]
--------------------------- (16)

21. Since we are imagining the case where Km > > [S] we neglect [S] in the
denominator and include the assumption that [Et] ≈ [E] since at very low [S]
relatively little [ES] should form:
v ≈
𝑘𝑐𝑎𝑡[E][S])
𝐾𝑚
= k ˈˈ [E][S] --------------------------- (17)
Once again, since kˈˈ = k cat/Km in eq 17, we refer to kcat/Km as an apparent
second order rate constant. Because kcat/Km is a measure of the rate of the
reaction divided by the term that reflects the steady-state affinity of the
enzyme for the substrate. It is considered an indicator of the catalytic
efficiency of the enzyme and sometimes is called the specificity constant.
To illustrate a upper limit value of k cat/Km , we rewrite kcat/Km as follows:

22. 𝑘𝑐𝑎𝑡
𝐾𝑚
𝑘3
(𝑘2 + 𝑘3)/𝑘1
=
--------------------------- (18)
𝑘𝑐𝑎𝑡
𝐾𝑚
𝑘3
(𝑘2 + 𝑘3)/𝑘1
=
Next, we imagine the case where k3 >> k2:
= k1
--------------------------- (19)
So we see that kcat/Km can approach k1 as a limiting value, and k1 is the
second-order rate constant for the productive collision of enzyme and
substrate and as such it is limited by diffusion to about 108 – 109 M-1 s-1 . Thus,
if we see an enzyme that has a kcat/Km value in the neighborhood of 108 – 109
M-1 s-1 we say that the enzyme has attained “catalytic perfection”. A number
of enzymes that catalyze “near equilibrium” reactions in metabolic pathways

23. Next, we return to eq 14
and consider what happens when v = ½ Vmax:
When we simplify eq 20 we find that Km = [S] (under the above conditions;
i.e., v = ½ Vmax). So, in other words, Km is formally defined as a collection of
rate constants (eq. 8), but it is also equal to the substrate concentration that
gives half-maximal velocity of the enzyme-catalyzed reaction.
𝑉𝑚𝑎𝑥
2
𝑉𝑚𝑎𝑥 [S]
𝐾𝑚 + [𝑆]
= --------------------------- (20)
1
𝑣
𝐾𝑚
𝑉𝑚𝑎𝑥
=
1
[𝑆]
1
𝑉𝑚𝑎𝑥
+ --------------------------- (21)

24. The equation (21) is the Lineweaver-Burk equation. When I/v is plotted
against 1/[S], a straight line is obtained. This line will have a slope of Km/Vmax,
an intercept of 1/Vmax on the 1/v axis and an intercept of -1/Km on the 1/[S]
axis.
1
𝑣
𝐾𝑚
𝑉𝑚𝑎𝑥
=
1
[𝑆]
1
𝑉𝑚𝑎𝑥
+ --------------------------- (21)
Slope=
1
𝑣
1
[𝑆]
𝐾𝑚
𝑉𝑚𝑎𝑥
1
𝑉𝑚𝑎𝑥
−1
Km

25. This plot is also known as double reciprocal plot.
Vmax can be determined from this plot.
Again if we rearranged the Michaelis- Mentend equation (eq. 21).
A plot of v against v/[S], called the
Eadie-Hofstee plot, not only gives
Vmax and Km in a very simple way
but also magnifies departures from
linearity which might not be
apparent in a double
reciprocal plot.
V= -Km .
𝑣
[𝑆] + Vmax
Slope=
𝑣
[𝑆]
𝑽𝒎𝒂𝒙
𝑲𝒎
-Km
v
Vmax

26. Lineweaver-Burk equation/ Double Reciprocal
equation
We know, for enzyme kinetics,
Michaelis Menten equation 𝑣=
𝑉𝑚𝑎𝑥 𝑆
𝐾𝑚+[𝑆]
Taking the reciprocal gives,
1
𝑣
=
𝐾𝑚+ 𝑆
𝑉𝑚𝑎𝑥 𝑆
𝑜𝑟,
1
𝑣
=
𝐾𝑚
𝑉𝑚𝑎𝑥
1
[𝑆]
+
1
𝑉𝑚𝑎𝑥
………………… (i)
Where, 𝑣= Reaction velocity ( the reaction rate)
Km = Michaelis-Menten constant
Vmax = Maximum reaction velocity
[S] = Substrate concentration
The equation (i) is called Lineweaver-Burk equation.

27. Eadie-Hofstee Plot
Michaelis Menten equation 𝑣 = 𝑉𝑚𝑎𝑥 𝑆
𝐾𝑚+[𝑆]
if we rearranged the Michaelis- Mentend equation, we will
get..
𝑣 = -Km .
𝑣
[𝑆]
+ Vmax
which not only gives Vmax and Km in a very simple way
but also magnifies departures from linearity which might
not be apparent in a double reciprocal plot.

28. Significance of Michaelis-Menten Constant:
(i) By knowing the Km value of a particular enzyme-substrate system, one can
predict whether the cell needs more enzymes or more substrate to speed
up the enzymatic reaction.
(ii) If an enzyme can catalyse a reaction with two similar substrates (e.g.,
glucose and fructose) in the cell, it will prefer that substrate for which the
enzyme has lower Km value.
(iii) Km value gives an approximate measure of the concentration of substrate
of the enzyme in that part of the cell where reaction is occurring. For
instance, those enzymes which catalyse reactions with relatively more
concentrated substrates (such as sucrose), usually have relatively high Km
value. On the other hand, the enzymes that react with substrates which
are present in very low concentrations (such as hormones) have
comparatively lower Km values for the substrates.

29. 29
What Affects Enzyme Activity?
(factors affect catalytic efficiency of enzyme)
• Three factors:
1. Environmental Conditions
2. Cofactors and Coenzymes
3. Enzyme Inhibitors

30. 30
1. Environmental Conditions
 1. Extreme Temperature are the most
dangerous
- high temps may denature (unfold) the
enzyme.
 2. pH (most like 6 - 8 pH near neutral)
 3. substrate concentration .

31. 31
2. Cofactors and Co-enzymes
• Inorganic substances (zinc, iron) and
vitamins (respectively) are sometimes need
for proper enzymatic activity.
• Example:
Iron must be present in the quaternary
structure - hemoglobin in order for it to pick
up oxygen.

32. Environmental factors
• Optimum temperature The temp at which enzymatic reaction occur
fastest.

33. Environmental factors
• pH also affects the rate of enzyme-substrate
complexes
• Most enzymes have an optimum pH of around 7
(neutral)
• However, some prefer acidic or basic conditions

35. Enzyme Inhibitors
• Competive - mimic substrate, may block active site, but may dislodge
it.

36. Enzyme Inhibitors
• Noncompetitive

37. 38
Catalytic Mechanism of Enzyme

38. 39
Catalytic Mechanism

39. 40
Types of Catalytic Mechanism

40. 41
1) Acid –base catalysis

41. 42
Concerted acid-base catalysis

42. 43
RNase A is an acid-base catalyst

43. 44
RNase A mechanism

44. 45

45. 46
2) Covalent catalysis

46. 47
Decarboxylation of acetoacetate

47. 48
Three stages of covalent catalysis

48. 49
Nucleophilicity of a substrate is
related to its basicity

49. 50
Important aspects of covalent
catalysis

50. 51
3) Metal Ion cofactors act as catalyst

51. 52
Metal Ion cofactors act as catalyst

52. 53

54. 55
4) Catalysis through proximation and
orientation effects

55. 56
4) Catalysis through proximation and
orientation effects

56. 57
Proximation and Orientation

57. 58
Geometry of an SN2 reaction

58. Identification of Functional
Groups Essential for Catalysis
Are some groups really necessary for enzyme catalysis?
1. Alkylation of ribonuclease treating with iodoacetate at pH 5.5
gives inactive alkylated ribonuclease. This is due to the
suppression of active catalytic sites by alkyl group.

59. The action of phosphorylating agent, diisopropyl
phosphofluoridate on hydroxyl group of serine residue
containing enzymes, e.g., chymotrypsin, trypsin.
This reagent is one of a group of toxic organophosphorus
compounds sometimes called nerve poisons because they
combine and completely inactivate the enzyme,
acetylcholinesterase which functions in the activity of nervous
system.
CH
CH2
OH
H
N C
O
Active serine residue of enzyme
+
CH3
HC
CH3
O
P CH
CH3
CH3
O
O
F
diisopropylphosphofluoridate

60. -HF
CH
CH2
O
H
N C
O
CH3
HC
CH3
O
P CH
CH3
CH3
O
O
Diisopropylphosphoric ester of enzyme

61. Acetylcholinesterase (AChE) is a hydrolase enzyme that
hydrolyzes choline esters. It catalyzes the breakdown of
acetylcholine and of some other choline esters that function as
neurotransmitters.
Acetylcholinesterase catalyzes the hydrolysis of acetylcholine to
acetic acid and choline.

62. Note: The functional groups of enzymes required for catalytic activity are usually
much more accessible or reactive than similar groups elsewhere in the molecule that
are not directly involved in catalysis.
For example, there are more many functional groups of ribonuclease to react with
iodoacetate but imidazole groups of Histidine residue 12 and His119 are far more
reactive than all the others.

63. Affinity labeling: The way of identifying essential functional
groups in enzyme active sites.
Affinity labels are molecules similar in structure to a
particular substrate for a specific enzyme and are considered to
be a class of enzyme inhibitors. The label binds covalently to
the enzyme so that the substrate can no longer bind, causing a
permanent and irreversible change.

64. Interconversion of
aldehyde and ethanol

65. Structured and reaction of Coenzyme NAD+

66. Redox reaction of NAD+

67. Alcohol- Aldehyde-Acid

68. After binding of NAD+ the water molecule is displaced from the zinc atom by the incoming
alcohol substrate. Deprotonation of the coordinated alcohol yields a zinc alkoxide
intermediate, which then undergoes hydride transfer to NAD+ to give the zinc-bound
aldehyde and NADH. A water molecule then displaces the aldehyde to regenerate the original
catalytic zinc centre, and finally NADH is released to complete the catalytic cycle.
Reaction Mechanism

69. Note: the role of zinc in the dehydrogenation reaction is to
promote deprotonation of the alcohol, thereby enhancing hydride transfer
from the zinc alkoxide intermediate. Conversely, in the reverse
hydrogenation reaction, its role is to enhance the electrophilicity of the
carbonyl carbon atom.

70. Alcohol dehydrogenases (ADHs) are a class of zinc enzymes
belonging to the group of oxidoreductases.
an oxidoreductase is an enzyme that catalyzes the transfer of
electrons from one molecule, the reductant, also called the
electron donor, to another, the oxidant, also called the electron
acceptor), which catalyze the reversible interconversion of
alcohols and the corresponding carbonyl compounds
(aldehydes or ketones) i.e., it is not only oxidizes ethanol to
acetaldehyde in animals but also produces ethanol from
acetaldehyde in yeast.

71. This interconvertion reaction is occurred by oxidation-reduction
reaction.
Ethanol NAD+ Acetaldehyde NADH
In the foreword reaction, alcohol dehydrogenase enzyme catalyses
the oxidation of ethanol to acetaldehyde by the transfer of a hydride
anion (H¯) to NAD+ with release of a proton. Here, the proton is
released into solution, while the ethanol is oxidized and NAD+
reduced to NADH by transfer of the hydride to the nicotinamide
ring.
NAD+ is oxidized and NADH is reduced form of Nicotinamide
adenine dinucleotide (NAD), which is a coenzyme found in all
living cells.

72. In metabolism (the chemical processes that occur within a living
organism in order to maintain life), nicotinamide adenine
dinucleotide is involved in redox reactions, carrying electrons from
one reaction to another. The coenzyme is, therefore, found in two
forms in cells: NAD+ is an oxidizing agent – it accepts electrons
from other molecules and becomes reduced. This reaction forms
NADH, which can then be used as a reducing agent to donate
electrons.
The redox reactions of nicotinamide adenine dinucleotide.

73. Enzymes are catalysts, that is, they must be
recovered at the end of the reaction and can only
speed up the rate at which everything comes to
equilibrium. So the dehydrogenases must catalyze
the reduction, that is the reverse reaction, in which
the strong reducing agent NADH reduces the
acetaldehyde to form ethanol and is oxidized to
NAD+.
This means the coenzyme can continuously cycle
between the NAD+ and NADH forms without being
consumed.

75. An esterase is a hydrolase enzyme that splits esters into an acid and an
alcohol in a chemical reaction with water called hydrolysis. A wide range
of different esterases exist that differ in their substrate specificity, their
protein structure, and their biological function.
Esterase Activity :
Enzyme-catalyzed interconversion of Ester and Carboxylic
acid

76. The principle of Microscopic Reversibility: For any reversible
reaction, the mechanism in the reverse direction must be identical
to that in the forward reaction (only reversed). This is a valuable
approach to study enzyme mechanisms.
Mechanism for acetylcholinesterase :

77. Like many hydrolytic enzymes, the reaction proceeds via a
covalent enzyme-substrate intermediate, formed when the acyl
group of acetylcholuine is initially transferred to an active-site
serine. A water nucleophile then attacks their ester, driving off
acetate (carboxylic acid) and completing the hydrolysis with
regenerating the free enzyme.