The document discusses enzymes as essential biocatalysts for biochemical reactions, emphasizing their roles in life processes, specificity, catalytic efficiency, and regulation. It covers enzyme structure, classification, and kinetics, including the Michaelis-Menten model, factors affecting reaction velocity, inhibition types, and regulatory mechanisms. Enzymes are vital to biological functions and are affected by various conditions such as substrate concentration, temperature, and pH.

1. Enzymes
Md. Saiful Islam
B.Pharm, M.Pharm (PCP)
North South University
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3. Enzymes
There are two fundamental conditions for life. First, the living entity must be able
to self-replicate; second, the organism must be able to catalyze chemical
reactions efficiently and selectively.
Life depends on the existence of powerful and specific catalysts which is known
as enzymes. Almost every biochemical reaction is catalyzed by an enzyme.
• Living procedures are based on chemical reactions and to accelerate
these reactions in cells require specific catalyst. Enzymes are proteins
which perform this function.
• Therefore, enzymes are biocatalyst produced by living cells to catalyze
biochemical reactions. A catalyst accelerate the rate of chemical reactions
but remains chemically unchanged at the end of reaction.
• Enzymes acted upon the metabolite is called the enzyme’s substrate.
• Without Enzyme there is no known live cells in the world

4. Enzymes needed in all the systems of the body, like
in the movement of nutrients for oxidation
in the conversion of energy from foods and
in the synthesis of macromolecules
in Blood circulation system
in the respiration system
in reproduction system

5. • Enzymes are Proteins except rybozyme (RNA Enzyme)
• Enzymes are 3 dimensional structure which is important for its activity
• Molecular weight of most of the enzymes are about 12kD to 1000kD
• Some are pure protein
• Most of the enzyme contains additional chemical structures other than
proteins, which are called cofactors
Enzyme Hormone
Absolutely protein in nature May be other than protein, eg, steroid
Act at the site of production Usually act distant to the site of
production
Transportation to the site of action
not via blood
Transportation to the target cell via blood
Shows catalytic activity Shows regulatory activity

6. 1. Active sites: Enzyme molecules contain a special pocket or cleft called
the active site. The active site contains amino acid side chains that create
a three dimentional surface complementary to the substrate. The active
site binds the substrate, forming the enzyme-substrate (ES) complex. ES
is converted to an Enzyme-Product (EP) complex that subsquently
dissociates to enzyme and product.
2. Catalytic efficiency: Enzymes are excellent catalysts, speeding up
reactions 105 to 1017 fold. They speed up reactions without being used up.
Typically each enzyme molecule capable to transfer 100-1000 substrate
molecules into product in each second.
3. Specificity: Enzymes are highly specific, interacting with one or few
substrates and catalyzing only one type of chemical reactions.
Since enzymes are extremely selective for their substrates, the set of
enzymes presence in a organelle of the cell determines which metabolic
pathways occur in that portion of the cell.
Fundamental Properties of Enzyme

8. 4. Regulation– Enzymes can be activated or inhibited, so that the
rate of product formation responds to the needs of the cell.
Some enzymes can be regulate metabolic signals.
5. Location within the cell: Many enzymes are localized in specific
organells within the cell. Such compartmentalization serves to
isolate the reaction substrate or product from other competing
reactions. This provides a favorable environment for the reaction
and organizes the thousands of enzymes present in the cell into
purposeful pathway.

9. 6. Holoenzyme:
In many cases, the enzyme consists of the protein and a combination of
one or more non-protein parts called cofactors. This enzyme complex is
usually simply referred as the holoenzyme.
Apoenzyme: The polypeptide or protein part of the enzyme is called the
apoenzyme and may be inactive in its original synthesized structure. The
inactive form of the apoenzyme is known as a proenzyme or zymogen.
The proenzyme may contain several extra amino acids in the protein
which are removed, and allows the final specific tertiary structure to be
an activated apoenzyme form.

10. Cofactors:
A cofactor is a non-protein substance which may be organic, and called
a coenzyme. The coenzyme is often derived from a vitamin.
Thiamine, B6, B12, NAD, FAD, Coenzyme A (CoA)
Another type of cofactor is an inorganic metal ion called a metal ion
activator. The inorganic metal ions may be bonded through coordinate
covalent bonds. The major reason for the nutritional requirement for
minerals is to supply such metal ions as Zn+2, Mg+2, Mn+2, Fe+2, Cu+2, K+1,
and Na+1 for use in enzymes as cofactors.

11. No Class Type of reaction catalyzed Examples
1 Oxidoreductases Transfer of electrons (hydride ions or H atoms) Dehydrogenases,
Oxidases
2 Transferases Group transfer reactions Transaminase,
kinases
3 Hydrolases Hydrolysis reactions (transfer of functional
groups to water)
Estrases,
Digestive
enzymes
4 Lyases Addition of groups to double bonds, or
formation of double bonds by removal of
groups
Decarboxylases,
Aldolases
5 Isomerases Transfer of groups within molecules to yield
isomeric forms
Fumerase, hexo-
isomerase
6 Ligases Formation of C-C, C-S, C-O, and C-N bonds by
condensation reactions
Citric acid
synthase
Classification of Enzymes

12. Enzyme Nomenclature
Recommended name:
The different kinds of enzymes are named in different ways:
• Most often enzymes are named by adding a suffix 'ase' to the root
word of the substrate. For example, Lipase (fat hydrolysing enzyme),
Sucrase (breaking down sucrose).
• Sometimes the enzymes are named on the basis of the reaction that
they catalyse. For example, Polymerase (aids in polymerisation),
Dehydrogenase (removal of H atoms).
• Some enzymes have been named based on the source from which
they were first identified. For example, Papayin from papaya.
• The names of some enzymes ends with an 'in' indicating that they
are basically proteins. For example, Pepsin, Trypsin etc.
Each enzyme has assigned for two names. The first one is it’s short,
recommended name, convenient for everyday use. The second is the
more complete systematic name, which is used when an enzyme must be
identified without ambiguity.

13. Systematic name:
IUBMB (International Union for Biochemistry and Molecular Biology)
developed a system of nemenclature in which enzymes are classified into
six major classes, each with numerous subgroup. The systematic names
are unambiguous and informative but are frequently too cumbersome to
be of general use.

14. • Six main enzyme groups are:
• EC 1 - Oxidoreductases
• EC 2 - Transferases
• EC 3 - Hydrolases
• EC 4 - Lyases
• EC 5 - Isomerases
• EC 6 - Ligases
• Enzymes are named and classified depending on the reaction
they catalyse.
• An EC number represents the reaction catalyzed by an enzyme
(protein) but not the enzyme (protein) itself. Therefore different
proteins can have the same EC number.
EC, Enzyme Commission

15. Example: (Glucose phosphotransferase, Hexokinase)
– E.C. Number: 2.7.1.1
• 2: transferase
• 7: subclass (phosphotransferase)
• 1: phosphotransferase with a hydroxyl group as acceptor
• 1: D-glucose as phosphoryl group acceptor
ATP + D-Glucose ADP + D-Glucose 6-phosphate
Hexokinase

16. EC 3. Hydrolases
EC 3.1 Hydrolases Acting on Ester Bonds
• EC 3.1.3 Phosphoric Monoester Hydrolases
– EC 3.1.3.1 alkaline phosphatase
– EC 3.1.3.2 acid phosphatase
– EC 3.1.3.3 phosphoserine phosphatase
– EC 3.1.3.4 phosphatidate phosphatase
– EC 3.1.3.5 5'-nucleotidase
– EC 3.1.3.6 3'-nucleotidase
– EC 3.1.3.7 3'(2'),5'-bisphosphate nucleotidase
– EC 3.1.3.8 3-phytase
– EC 3.1.3.9 glucose-6-phosphatase
– EC 3.1.3.10 glucose-1-phosphatase
– EC 3.1.3.11 fructose-bisphosphatase

17. Factors afecting reaction velocity:
Enzymes can be isolated from cells and their
properties studied in vitro.
Velocity: The rate or velocity of a reaction is the
number of substrate molecules converted to
product per unit time, velocity is usually
expressed as µmol of product formed per
minute.
Factors affecting reaction velocity:
1. Substrate concentration:
The rate of an enzyme-catalyzed reaction
increases with increasing substrate
concentration until a maximal velocity is
reached. The plot of initial reaction velocity
against substrate concentration is hyperbolic.
[S]
Rate

18. 2. Temperature:
The reaction velocity increases with temperature until a peak velocity is
reached, and further elevation of the temperature results in a decrease in
reaction velocity because of the temperature-induced denaturation of the
enzyme.
Optimum temperature of human enzyme : 35-40°C
>40°C denaturation starts
Thermipholic bacteria in hot spring can survive at approx 70°C
Relationship of velocity to enzyme concentrations: The rate of the
reaction is directly proportional to the enzyme concentration at all substrate
concentrations. For example, if the enzyme concentration is reduceed by
50%, the initial rate of reaction (V0), as well as that of Vmax are reduced to
half of the original.

19. 3. pH (acid-base property):
[H+] affects reaction velocity
The catalytic process usually requires specific chemical groups (ionized or un-
ionized) in enzyme or substrate in order to interact.
eg, if a catalytic activity requires an amino group of an enzyme to be a
protonated form (-NH3
+), at alkaline pH this group is deprotonated and
therefore the reaction velocity declines.
Extremes of pH may leads to denaturation of the enzyme because the
structure of the catalytically active protein molecule depends on the ionic
character of the amino acid side chains.
Optimum pH varies for different enzymes: eg pepsin (digestive enzyme in
stomach) maximally active at pH 2.0, whereas enzymes, supposed to work at
neutral pH are denatured by such an acidic environment.

20. Reaction Rates and the Transition State
Enzymes speed up reactions enormously.
To understand how they do this, examine the concepts
of activation energy & the transition state.
In order to react, the molecules involved are forced to
have an unlikely electronic arrangement
To proceed the reactions the molecules must pass
through a high energy state.

21. This high energy state is called the transition state.
The energy required to achieve the transition state for
one mol of the substrate is called the activation
energy for the reaction.
The higher the activation
energy for the transition
barrier, the slower the
reaction rate.

22. Enzymes lower energy
barrier by forcing the
reacting molecules
through a different
transition state.
This transition state
Involves interactions
with the enzyme.
Enzyme

23. Enzyme Kinetics
Kinetics Kinetics refers to the rate of change in a
biochemical (or other) reaction i.e. the study of reaction rates.
Kinetic comes from the Greek word "kinesis" which means
motion.
Enzyme Kinetics Rate of chemical reactions mediated by
enzymes. Enzymes can increase reaction rate by favoring or
enabling a different reaction pathway with a lower activation
energy, making it easier for the reaction to occur.

24. Michaelis-Menten Model or Equation
k1 k3
E + S  E●S  E+ P
k2
From this kinetic scheme, a relationship can be
derived for the rate or velocity of the reaction:
Michaelis-Menten Equation
Vmax[S]
[S] + Km
V =

25. v or
rate
[S]
Vmax
Km
½ Vmax
0
Vmax, the maximum rate (plateau)
is k3 x [total enzyme]
Km =(k1 + k3)/ k2, almost a
binding constant
Michaelis-Menten constant
(Km) can be defined as the
concentration of the specific
substrate at which a given
enzyme reaches one-half its
maximum velocity.
Km = [S], where the velocity v
= ½ Vmax,
Km is called the Michaelis
constant.
k1 k3
E + S  E●S  E+ P
k2

26. Km reflects the affinity of the
enzyme for a substrate.
•Low Km value reflects a high affinity
of the enzyme for substrate,
because a low concentration of
substrate is needed to half-saturate
the enzyme that is to reach the
velocity at ½ Vmax.
•High Km reflects the low affinity
enzyme for substrate because a
high concentration of substrate is
needed to half-saturate the enzyme.
v
Vmax/2
Km
[S]
Vmax

27. Lineweaver-Burk plot:
This form of the Michaelis-Menten equation is
called the Lineweaver-Burk equation. For
enzymes obeying the Michaelis-Menten
relationship, a plot of 1/V0 versus 1/[S] yields a
straight line. This line has a slope of Km/Vmax, an
intercept of 1/Vmax on the 1/V0 axis, and an
intercept of 1/Km on the 1/[S] axis. The double-
reciprocal presentation, also called a Lineweaver-
Burk plot, has the great advantage of allowing a
more accurate determination of Vmax, which can
only be approximated from a simple plot of V0
versus [S].

28. Each enzyme has a characteristic Km for a given substrate
Enzyme Substrate Km, mM
Catalase H2O2 25
Hexokinase ATP 0.4
D-Glucose 0.05
D-Fructose 1.5
-Galactosidase D-Lactose 4.0
Threonine dehydratase L-Threonine 5.0

29. Enzyme measurement:
Unit: One unit of enzyme activity is defined as that amount
causing transformation of 1µmole of substrate to product per
minute at 25°C under optimal conditions of measurement.
Unit of enzyme is expressed as μmoles/min/mg protein
Specific activity:
The specific activity is the number of enzyme units per mg of
protein. The specific activity is a measure of enzyme purity.

30. Inhibition of Enzyme activity
• Substance that can diminish the velocity of an enzyme-catalyzed
reaction is called an inhibitor.
Two types of Enzyme Inhibition:
• Reversible inhibitors bind to enzymes through non-covalent
bonds, thus dilution of the enzyme-inhibitor complex results in
dissociation of the reversibly bound inhibitor.
• Irreversible inhibitors bind to enzymes through covalent bonds.
Reversible inhibition:
Competitive inhibition, Noncompetitive inhibition

31. I (inhibitor) resembles S (substrate)
I binds at active site reversibly
E●I cannot bind to S so no reaction
Competitive inhibition: Occurs when the inhibitor binds reversibly to the
same site that the substrate would normally occupy and, therefore,
competes with the substrate for that site.

32. No I
+I
+more I
Vmax
Km
In competitive inhibition, addition of enough
substrate can overcome the inhibition.
 same Vmax

33. Effect of competitive inhibitor on Vmax:
The effect of a competitive inhibitor is reversed by increasing [S]. At a
sufficient high substrate concentration, the reaction velocity reaches
the Vmax as observed in the absence of inhibitor.
Effect of competitive inhibitor on Km:
A competitive inhibitor increases the apparent Km for a given substrate
which means that, in the presence of a cmpetitive inhibitor, more
substrate is needed to achieve ½ Vmax.
Statin drugs: competitively inhibits HMG-CoA reductase (rate
limiting enzyme) and thus inhibit synthesis of cholesterol, thereby
lowering plasma cholesterol levels.

34. Noncompetitive inhibition:
Occurs when the substrate and inhibitor bind at different sites
on the enzyme, binds either free enzyme or the ES complex,
thereby preventing the reaction.
E + S  E●S E + P
+ +
I I
 
E●I  E●S●l
inhibitor
substrate
Inhibitor binds the enzyme somewhere
different from where the substrate binds.
So the inhibitor does not care whether
substrate is bound or not.
Inhibitor changes the conformation of
the enzyme at the active site so reaction
is not possible with inhibitor bound.

35. Effect on Vmax:
Noncompetitive inhibition can not be overcome by increasing the
concentration of substrate, thus decrease the apparent Vmax of the
reaction.
Effect on Km:
Noncompetitive inhibitors do not interfere with the binding of
substrate to enzyme, thus the enzyme shows the same Km in the
presence or absence of noncompetitive inhibitor.
eg:
Pb noncompetitively binds with Ferrochelatase and inhibits
insertion of iron into protoporphyrin (a precursor of heme).

36. Irreversible Inhibition
combine covalently to enzyme so as
to permanently inactivate it
(previous examples are all reversible)
almost all are very toxic
most bind to a functional group in
active site of enzyme to block that site

37. Example 1:
Diisopropyl fluorophosphate (DFP) binds covalently with
serine proteases and acetylcholinesterase – used as biological
weapon.
Sarin (DFP derivative) is a deadly nerve gas  Paralysed in
certain functions because of the failure of nerve impulses to be
transmitted properly.
DFP + Acetylcholinesterase = Diisopropylphosphate ester of
cholinesterase (inactive) + HF
Once acetyl choline has been secreted it binds to the receptor sites to
the next nerve cells to propagate the nerve impulses. Before the
second impulse, acetylcholine must be hydrolysed by
acetylcholinesterase in the junction. The irreversible inhibitor DFP is
very reactive and combines with acetylcholinesterase and the
derivative has no longer functional capacity.

38. Example 2:
penicillin and related antibiotics bind covalently
to a peptidase involved in cell wall synthesis in
bacteria
Staphylococci, Streptococci sp.

39. Regulatory Enzymes
Enzymes whose activity is modulated through various types
of molecular signals, are called regulatory enzymes.
Two major classes of regulatory enzymes:
• Allosteric or noncovalently regulated enzymes
• Covalently regulated enzymes

40. Allosteric enzymes: The term allosteric derives from Greek
word which means other sites, that is allosteric enzymes are
those having other sites.
Like all enzymes, allosteric enzymes have catalytic sites, which
bind the substrate and transform it, but they also have one or
more allosteric sites for noncovalent binding of regulatory
compounds which are generally small metabolites or cofactors.
The allosteric site is specific for its modulator, allosteric enzyme
molecules are generally larger and more complex than those of
simple enzymes, most of them have two or more polypeptide
chains or subunits, allosteric enzymes usually show significant
deviations from classical Michaelis-Menten behavior.

42. Kinetics of Allosteric enzymes:
Allosteric enzymes show relationships between velocity and substrate
concentrations that differ from Michaelis-Menten kinetics. Allosteric enzyme
shows sigmoidal curve rather than hyperbolic curves like all other non-
regulatory enzymes. Sigmoid kinetic behavior generally reflects cooperative
interactions between protein subunits.
Enzyme

43. Enzymes in clinical diagnosis
Plasma enzymes: There are two types of plasma enzymes
a) Relatively small group of enzymes are actively secreted into
the blood by certain cell types for specific functions.
b) A large number of enzymes are released from cells during
normal cell turnover. These enzymes normally functions
intracellularly and have no physiologic use in plasma. In
healthy individuals these enzymes are fairly constant.
Elevation of these enzymes in plasma indicates tissue
damage.

44. C. Increased levels of ALT/GPT indicates hepatic tissue damage,
Normal range : up to 40 U/L
Creatin kinase (CK): two subunit, M and B,
Three isozymes: CK1=BB, CK2=MB, CK3=MM
Myocardial muscle is the only tissue that contains CKMB, more
than 5% of total CK is CKMB,
In acute myocardial infarction, CKMB elevated in 4 to 8hrs, max
in 24 hrs, returns to normal in 48 to 72hrs,
Range: 25 U/L
Troponin I: highly specific for myocardial infarction, appears in
plasma within 4 to 6 hrs, max in 8 to 28hrs, remain elevated for 3
to 10days,
Range: normal <0.1ng/ml, 0.1-0.25 intermediate, >0.25ng/ml
moderate to severe

45. Enzymes may catalytically defective due to genetic
mutation
Disease Defective Enzyme
Albinism Tyrosine 3-monooxygenase
Alkaptonuria Homogentisate 1,2-dioxigenase
Galactosemia Galactose 1-PO4 uridylyl transferase
Phenylketonuria Phenylalanine 4-monooxigenase