Enzymes
Enzymatic Activity and Catalysis:
ΔG is the free energy of the overall reaction and it is
independent of the enzyme and is a constant for
every reaction.
Enzymes decrease the activation energy of reactions.
Units of Measurement of Enzymes:
1- Standard Unit of Enzyme Activity (U)  It is the
amount of enzyme that reacts with one
micromole of the substrate per minute
(mMole/min).
2- Specific Activity of the Enzyme  it is an
expression of the amount of enzyme activity
units (U) per milligram of the enzyme.
3- Catalytic Constant of the Enzyme  it is an expression of the amount of enzyme activity units (U) per mole of
the enzyme.
The Enzyme-Substrate Complex
The Active Site of the enzyme:
1- Definition  it is the region of the enzyme to which the substrate binds.
2- Composition  the active site is composed of the functional groups of some amino acids. The 3-dimensional
structure of these functional groups and their proximity allows them to bind to the substrate.
The 3-dimensional arrangement of the enzymes are so important because if the arrangement is lost, the enzyme does
not bind to the substrate. For example, Lysozyme in its 1ry structure has no activity.
The substrates are bound to the enzymes by WEAK attraction forces, which are NON-COVALENT bonds (e.g. ionic
attractions and van der waal forces)
The enzyme and the substrate should have complementary shapes.
Theories explaining the binding of enzymes and their substrates:
1- Key and lock theory (Fischer or Template model):
Postulate  the active site of the enzyme has a fixed shape (i.e. the substrate is pre-shaped before the substrate
binding).
Shortcomings (Disadvantages)  this theory could not explain why other molecules than the substrate can bind to
certain enzymes, which suggest that the enzymes do NOT have a fixed shape.
2- The Induced-fit model of Koshland:
Postulate  the active site of the enzymes have shapes that are complementary to that of the substrate only after the
binding of the substrate (i.e. the enzyme is NOT pre-shaped.)
Mechanism  the substrate induces the active site.
Advantages  It explains how more than one substrate can bind to an enzyme.
The Specificity of Enzymes:
Enzymes are highly specific both in the reaction catalyzed and their choice of substrates.
The specificity is determined by  the functional groups of the enzyme, cofactors & the substrates and the physical
proximity between these groups.
Types of Enzyme specificity:
1- Relative Specificity  the enzyme works on many substrates but has a ↑ affinity for a specific substrate.
-
Examples  Lipases and Proteases.
2- Structural (positional) Specificity  the enzyme works on a specific bond in a very specific position.
-
Examples  Pepsin (works between the carbonyl of aspartic acid and the Nitrogen of tyrosine)
3- Absolute Specificity  the enzyme works on only one substrate.
-
Examples  Ureases & Sucrase
4- Optical Specificity  the enzyme works on only one substrate in a specific stereo form.
-
Examples  Glycolytic enzymes & amino acid oxidases.
Enzyme Kinetics
Factors affecting enzymatic activity (measuring enzymatic activity):
1- Enzyme Concentration:
2- Substrate Concentration:
-
At ↓ concentrations of the substrate,  the reaction has a first-order reaction kinetics.
At ↑ concentrations of the substrate,  the reaction has a zero-order reaction kinetics.
Km (Michaelis constant) Value:
-
Definition  it is the amount of substrate needed for the reaction rate to reach ½ Vmax.
Characters 
Km is inversely proportional to the affinity of the enzyme to the substrate.
Km is independent of the enzyme concentration.
If an enzyme acts on several substrates, it will have more than one Km for each substrate.
Michaelis-Menten Equation:
Lineweaver-Burk Plot:
Uses  Determination of Km and VMAX & Studying the mechanism of action of Enzyme Inhibitors.
Graph and Formula 
N.B. The value of VMAX reflects the amount of active enzyme present and it is independent of the substrate’s
concentration.
3- Temperature:
At ↓ temperatures  Energy < Eact
At ↑ temperatures  Denaturation due to cross-links destruction.
Enzymes need very specific temperatures (usually 37oC)
4- pH:
Effective concentration of an enzyme (or a substrate)  It is the concentration of the enzyme that can actually bind to
the substrate (has the required 3-D shape and charge).
The pH of the medium causes alterations in the charges of both the Enzyme and the substrate, which changes their
effective concentrations.
Enzyme denaturation occurs at high pH.
5- Time of the reaction & Product of the reaction
6- Light and other physical factors
Coenzymes
Apoenzyme + Coenzyme = Holoenzyme
Apoenzyme  Determines the substrate. It’s the protein part that connects to the substrate.
Coenzyme  Determines the biochemical reaction. It accepts or donates groups removed or added to the substrates,
thus is regarded as 2ry substrate sometimes.
Some of the coenzymes are vitamin B derivatives and are of nucleotide structure.
Coenzymes are classified according to the group they transfer.
Enzyme
Coenzyme
NAD
+
NADP
Dehydrogenases
FAD
FMN
Lipoic Acid
Pyruvate
Dehydrogenase
Amino
Transferase
(Trans-aminase
Reactions)
Ligases
(Carboxylases)
Transferase
TPP (Thiamine
pyrophosphate)
Group
Transferred
Hydrogen
(As HYDRIDE)
Coenzyme Reaction
Hydrogen
(As HYDRIDE)
Hydrogen
(As ATOM)
Hydrogen
(As ATOM)
Hydrogen
NADP + 2H 
NADPH + H+
FAD + AH2  FADH2
+A
Vitamin
Precursor
NAD++ AH2 
NADH + H+ + A
+
Niacin (Vit
B3)
Riboflavin
(Vit B2)
S—S  SH , SH
PLP (Pyridoxal
Phosphate)
Pyridoxine
(Vit B6)
Biotin
CO2
Vitamin H
Acyl group
Nicotinamide + Adenine +
Dinucleotide (2 Ribose + 2
Phosphate)
Nicotinamide + Adenine +
Dinucleotide Phosphate
Flavin + Adenine +
Dinucleotide
Flavin + Mono Nucleotide
6,8-dithio-octanoic acid
Aldehyde
(Acetal)
Aldehyde
(Acetal) 
Amino Group
Co-enzyme A
Structure Components
Thiamin
(Vit B1)
Pantothenic
Acid (Vit B5)
Thiamine + 2 Phosphate
groups
Pyridoxine (Vit B6) +
Phosphate
Nitrogenous base + Sugar
+ P + P + Pantothenic Acid
(Has a thioethanol amine
which is the active
moiety)
Inhibition of Enzyme Activity
An Inhibitor  is any substance that can diminish the velocity of an enzyme-catalyzed reaction.
Enzyme Inhibitors
Reversible Inhibitors
Competitive
Inhibitors
Uncompetitive
Inhibitors
Irreversible Inhibitors
Non-competitive
Inhibitors
Significance of Enzyme Inhibition Study:
1- Enzyme inhibition is a major control mechanism in the biological systems.
2- Many drugs and toxic chemicals act by inhibiting enzymes.
3- It is useful in studying the mechanism of enzyme action.
Group-Specific Irreversible Inhibitors:
1Irreversible Inhibitor  DIPF (Diisopropyl fluorophosphates)
Enzyme  Acetylcholinesterase
It reacts with a critical Serine (-OH) group.
2Irreversible Inhibitor  Iodoacetamide
Enzyme  Enzymes containing (-SH) group of cysteine.
It reacts with a critical cysteine (-SH) residue.
Competitive
Non-competitive
(Enzyme Poison)
Reversible Inhibitors
Competitive Inhibitors
Uncompetitive
Inhibitors
Mechanism
The inhibitor resembles the
substrate and binds at active site
The inhibitor binds to the ES
complex
Non-Competitive
Inhibition
The Inhibitor and the
substrate bind on different
sites on the enzyme. The
inhibitor can bind to
Enzyme or ES complex.
Diagram for
the
Mechanism
Reaction
Equation
Overcome by
↑↑ Substrate Concentration
Dialysis via semipermeable
membrane
Direct Plots
LineweaverBurk Plot
Effect
on
Enzym
e
Kinetic
s
Km
Vmax
↑↑
No change [G.R.]  Available
amount of enzyme has not
changed. If [S] is ↑↑ then Vmax
can be reached.
↓↓
No Change
↓↓
↓↓ [G.R.]  because the
number of active enzymes
is decreased.
Competitive Inhibitors
Examples
1C.I.  Malonate
Enzyme  Succinase Dehydrogenase
Substrate  Succinate
2Sulfa drugs (Sulfanilamide) is a C.I. for PABA, which is
required by M.O. for folic acid synthesis.
N.B. Sulfa drugs do not harm humans [G.R.]  Humans
cannot produce folate.
3Folate Analogs (e.g. Aminopterin, Methotrexate) acts as
antimetabolites. They are used as anti-tumor agents in
Childhood leukemia.
C.I.  Methotrexate and Aminopterin
Enzyme  Dihydrofolate Reductase
Substrate  Folic Acid
N.B: Methotrexate bounds 1000 fold better than folate to
DHF reductase.
S.E. of Methotrexate  Bone marrow suppression, GIT
mucosal damage and drug resistance.
4Lovastatin Is a hypercholesteremic drug that inhibits
endogenous cholesterol synthesis.
C.I. –> Lovastatin
Enzyme  HMG Co-A Reductase
Substrate  HMG Co-A (Hydroxymethylglutyryl Co-A)
Uncompetitive
Inhibitors
NonCompetitiv
e Inhibition
Extract of the
parasite
(Ascaris)
secretes pepsin
and trypsin
inhibitors so
that the
parasite
escapes
digestion in
intestine.