Enzymes are biological catalysts that are essential for life. They catalyze biochemical reactions efficiently and selectively. Enzymes lower the activation energy of reactions, increasing their rate. Most enzymes are proteins that use their tertiary structure and amino acid residues within their active site to catalyze reactions. The active site facilitates reactions by bringing substrates close together, stabilizing transition states, and using mechanisms like acid-base catalysis. This allows reactions to proceed rapidly under mild biological conditions. Without enzymes, reactions in living organisms would not occur at a useful pace to sustain life.
4. Introduction
• There are two fundamental conditions for life
– Organisms must be able to self-replicate
– Organisms must be able to catalyze chemical reactions
efficiently and selectively
• Without catalysis chemical reactions like glucose
oxidation couldn’t occur at a useful pace to sustain
life
• Enzymes are central to every biochemical process.
Acting in organized sequences they catalyze the
hundreds of stepwise rxns that occur to sustain life
5. Introduction
• With the exception of a few catalytic RNA
molecules (ribozymes), all enzymes are proteins
– Some enzymes require cofactors (e.g. inorganic ions,
coenzymes)
• Their catalytic activity depends on the integrity of
their native protein conformation
• The primary, secondary, tertiary and quaternary
structures of protein enzymes are essential to their
catalytic activity
6. Enzymes and Life
• The living cell is the site of numerous
biochemical activities - metabolism
• A site of processes of chemical and physical
change which goes on continually in the living
organism
7. Enzymes and Life
• Building of new tissue, replacement of old
tissue, conversion of food to energy, disposal
of waste materials are activities that
characterize LIFE
• Most of these activities are enzyme catalyzed
making enzymes crucial to life
8.
9. Enzymes and Life
• The enzymatic catalysis of biological reactions is
essential to life
• The normal biological conditions of living
organisms/cells. Such as; neutral pH, mild
temperature, aqueous environment; would not favor
chemical reactions at a useful rate without the aid of
catalysis
• Enzymes get around this problem (of mild
conditions) by providing a specific environment that
enables a given reaction to occur more rapidly
10. Enzymes and Health
• Physiological Roles in vivo
• Breakdown of nutrients to supply energy and simple
biomolecules (building blocks) - Catabolism
• The synthesis of proteins, polysaccharides, lipids,
DNA, membranes, cells, tissues, etc. - Anabolism
• The harnessing of energy to power cell motility,
transmembrane transportation, neural function,
muscle contraction, etc.
12. Enzymes and Health
• Disorder in enzyme quantity or functioning causes
disease
• Changes in the quantity or in the catalytic activity of
key enzymes that can result from
– genetic defects, nutritional deficits, tissue damage,
toxins, or infection by viral or bacterial pathogens
• Various treatments and pharmacologic agents
address disease conditions by altering activity (or
quantity) of enzymes (of patients or microbes)
– Anti hypertensives, anti acids, Antibiotics, ARVs, etc.
13. Other Uses of Enzymes
• Diagnostics
–ELISA - serum Abs, food allergen, detecting
antigens eg. pregnancy hormones
–Assaying specific enzymes in plasma as proxy for
tissue damage (ALAT, CK, LDH, etc.)
–The catalytic activity of enzymes reveals their
qualitative and quantitative presence in various
mixtures
14.
15. Other Uses of Enzymes
• Food industry
–Lactase to deplete lactose in foods for lactose
intolerants
–Brewing, baking, predigest baby foods, cheese
making
• Soap and detergent making
16. Enzymes
• Are substances that increase rates of a
chemical reactions,
–Usually by at least 106 times
• Enzymes are biological catalysts and most
are proteins EXCEPT for few RNAs with
catalytic activity called Ribozymes
17. Enzymes
• Catalyze the biological reactions that are
energetically possible (thermodynamically
favourable)
• Do not affect equilibrium position of the
reaction
• Does not affect the change in Free Energy
18. Enzymes
• Exhibit selectivity and specificity
–Type of reaction catalysed
–Type of bond synthesized or broken
• Different glycosidic bonds for eg.
–Substrate/substrate group
–Stereospecificity – 3D substrate-enzyme
interaction
• D vs L- sugars and amino acids, etc.
19. Enzymes
• The remarkable specificity of enzyme activity
affords living cells the ability to
simultaneously conduct many diverse
biochemical processes and control them
independently
20. Because enzymes
are extremely
selective for their
substrates,
-the set of enzymes
made in a cell
determines which
metabolic pathways
occur in that cell.
21. Definitions
Enzymology: is the study of enzymes
Catalyst : a substance that increases the rate of
a reaction without itself being consumed
(changed)
- Enzymes: are biological catalysts
22. Definitions …
• Cofactors – Non-protein organic
compounds/ inorganic ions needed for
enzyme’s catalytic activity (Helpers)
–Coenzymes (complex organic molecules)
–Inorganic ions (metallic cofactors)
• In nutrition the list of essential trace elements reflects
their role as cofactors, Fe2+/3+, Mn2+, Mg2+, Zn2+, Cu,
Co, etc.
23. Definitions …
• Coenzyme – a complex organic or
metalloorganic molecule (often B vitamin
derivatives) that is loosely associating with
and needed to make an enzyme catalytically
active (Dissociable carrier) (cosubstrate)
• Prosthetic group – coenzyme, metal ion or
metalloorganic molecule that is very tightly
bound (sometimes covalently) to a protein
24. Definitions …
• Holoenzyme – enzyme with a cofactor
• Apoenzyme – the polypeptide part of a
holoenzyme (protein alone)
• In enzyme catalyzed reactions, reactants are
referred to as substrates (S)
25. Enzyme Catalysis
• Enzymes carry out catalysis by accelerating
chemical reactions without themselves undergoing
permanent chemical change
• Enzymes are responsible for bringing about almost
all of the chemical reactions in living organisms
• Without enzymes, these reactions take place at a
very slow rate and would not meet the reaction
rates required for metabolism
26. Enzyme Catalysis
• An enzyme catalysed reaction occurs within a
catalytic pocket/site on the enzyme called the
Active site
• Active site of an enzyme molecule contains amino
acid side chains that create a 3D surface
complimentary to the substrate
• The side chains of the amino acid residues that line
the surface of the active site (with/without a
cofactor) interact with the substrate and
catalyze/facilitate its chemical transformation
28. • General features of active sites:
–The active site takes up a relatively small
portion of the total enzyme volume
–The active site is a 3 dimensional (3D) entity
or unit or station or location of the protein
enzyme
• Environment that shields substrates from
solvent
• Facilitates catalysis, bring molecules in close
proximity and/or in alignment or orientation
that favors reaction
29. –The specificity of binding depends on the
precisely defined arrangement of atoms in
the active site giving a direct fit or an
induced fit.
• Shape and bonding interactions btn active site
and substrate
30. • General features of active sites:
–Most substrates are bound to enzymes by
relatively weak forces
–Active sites are clefts or crevices formed by
folding of the polypeptide chain into a specific
shape
–Mostly, polar amino acids like Cys, Ser, Thr, Tyr,
His are found at the active site
31. Enzyme Catalysis
• The reaction to dissolve CO₂ in water to obtain
carbonic acid is very slow when not catalyzed
• In the body this reaction is catalysed by carbonic
anhydrase. The enzyme raises the rate of this
interconversion 10 million times
32. Enzyme Catalysis
• During the reaction each enzyme molecule
combines with its substrates, CO₂ and H₂O and
converts them to H₂CO₃. The enzyme and product
then separate and the enzyme can bind another
substrate.
• One molecule of the enzyme can hydrate 100,000
molecules of CO₂ per second.
33. Characteristics of enzyme catalysis
• Enzymes act to increase the rate of a reaction
• Enzymes do not affect the equilibria of
chemical reactions.
34. Characteristics of enzyme catalysis
• Many reactions in the body can go in either
direction. Indicated by ↔ in chemical
equations
• The direction of chemical reaction is not a
function of enzyme activity but depends on
the free energy content and concentration of
reactant and product molecules
35. Characteristics of enzyme catalysis
• Enzyme and substrate combine to form an
activated complex called an enzyme-substrate
complex (ES).
• This complex then undergoes a chemical
change to form product(s) and regenerates
the enzyme.
36. Characteristics of enzyme catalysis
• Enzymes are far superior catalysts than their
laboratory, non-biological counterparts.
–Enzymes have enormous Catalytic power -
affinity, dynamism
–They are highly specific
–Their activity can be regulated
37. Theories explaining enzyme-substrate
Interaction
• Lock and Key Theory
– The specific action of an enzyme with its substrate can
be explained using a Lock and Key analogy
– This theory was first postulated in 1894 by Emil Fischer.
– Useful in describing the discriminating specificity with
which enzymes recognize and interact with their
substrates
• Only the correctly sized/shaped key (substrate) fits into the
key hole (active site) of the lock (enzyme).
– Good in analogizing specificity of interaction but the
implied rigidity of enzyme active site is misleading
38. Theories explaining enzyme-substrate
Interaction
• Induced fit model of the active site
– Substrates induce a conformational change of the active
site as they approach and bind enzymes
• Hand into a glove analogy
– In the induced fit model of enzyme-substrate binding,
the shape of the active site of the unbound enzyme is
not the exact complement of the shape of the substrate.
– There is reciprocal changes induced to the substrate by
the enzyme which facilitate the intended transformation
of substrates to products – Binding energy
39. Mechanism of enzyme action
• The formation of an enzyme-substrate complex is
the first and crucial step in enzyme catalysis
• Substrate and enzyme interact over only a small
region of the enzyme surface, the active site.
• The enzyme-substrate complex formation
accelerates the inter-conversion of substrates to
products
40. Mechanism of enzyme action
• The mechanism of enzyme action can be
explained from two associated perspectives
–Energy perspective
• Enzymes provide an alternate, lower activation
energy, reaction pathway different from the
uncatalyzed reaction.
–Physico-chemical influence of the active site on
substrate-product conversion (catalysis)
41. Mechanism of enzyme action
I. Energy changes during a reaction
–Virtually all chemical reactions proceed by
overcoming an energy barrier separating
reactants and products. The free energy of
activation, ΔGEa
42. Mechanism of enzyme action
I. Energy changes during a reaction
–Free energy of activation is the energy difference
between that of reactants and a high-energy
intermediate (transition state) formed during the
formation of a product
–Because of high activation energy, the rates of
uncatalyzed chemical reactions are often slow.
• Relationship between ΔGEa and reaction rate is
inverse and exponential
43. Mechanism of enzyme action
–Activation energies are energy barriers
to chemical reactions
–These barriers may be accounted for by
• Bonds that need to be broken in reactants
• Slim chance of unassisted correct orientation
of reactant interaction
• Interference by other chemical groups in the
medium, etc
44. Mechanism of enzyme action
–The rate at which a molecule undergoes a
particular reaction decreases as the
activation barrier for that reaction increases
45. Mechanism of enzyme action
–These barriers are important to life
• Without such barriers, complex
macromolocules would spontaneously go back
to much simpler molecular forms and affect
the existence of the complex and highly
ordered structures and metabolic processes of
cells
• Protein, DNA, triglycerides do not
spontaneously disintegrate to respective
monomers(residues) although that is
thermodynamically favourable
46.
47. Mechanism of enzyme action
–In the absence of an enzyme only a small
proportion of a population of molecules may
posses enough energy to overcome the energy
barrier of the transition state.
–The rate of reaction is determined by the number
of such energized molecules.
48. Mechanism of enzyme action
–The lower the free energy of activation, the
,more molecules have sufficient energy to pass
through the transition state, and the faster the
reaction.
–An enzyme provides an alternate pathway with a
lower free energy of activation allowing the
reaction to proceed rapidly
50. Mechanism of enzyme action
• Factors contributing to the Activation energy
include:
– High entropy of free reactants
– Solvation shell that stabilizes free reactants
– Bond rearrangement that needs to occur
51. Mechanism of enzyme action
• Enzymes interact chemically with substrates
through the active site and by so doing overcome
the above factors
– Introduce entropy reduction
– The active site catalytic group interacting with substrate
desolvates the substrate
– The covalent and non covalent weak interactions btn
substrate and functional groups in the active site
initiates electron redistribution and set in motion the
bond rearrangements
52. Mechanism of enzyme action
• Enzyme-substrate interaction forms a stable
complex
• Stabilisation by these interactions is associated with
a release of free energy, the binding energy
• Binding energy contributes to lowering/countering
the activation energy and is a crucial source of
catalytic power of enzymes
• Interactions in this complex are optimized with the
substrate in its transition state
53. Mechanisms of enzyme action
II. Chemical and physical influence of the active
site
• Catalysis by proximity
–Bringing reactants to within bond-forming
distances of each other
–By binding the active site, reactants are brought
closer, in ideal spatial orientation and in an
environment that largely excludes other
interfering chemical groups present in medium,
hence promoting their reaction
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57. Mechanisms of enzyme action
• Catalysis by strain
– Enzymes that catalyze lytic reactions
– Typically bind substrates in way that strains the bond to
be broken/cleaved
– By binding the substrate in such a way that is
unfavorable to the bond that is to be cleaved the
enzyme helps that substrate approach its transition
state, hence the concept of transition state stabilization
59. Mechanism of enzyme action
–Transition state stabilisation: the active site
binds the substrate in such a way it resembles the
activated transition state. This greatly increases
the concentration of reactive intermediates ready
to be converted to product.
60. Effect of enzyme on the activation
energy
• Stabilization of the transition state
61. Mechanisms of enzyme action
• Acid-Base Catalysis
– Ionizable fuctional groups from side chains of aminoacyl
residues (of the enzyme polypeptide) lining the active
site, coenzymes, metal cofactors, and/or prosthetic
groups act as acids and bases.
– Facilitate catalysis through taking part in proton transfers
that aid chemical reactions
– Partly explains why rates of enzymatic reactions are
affected by changes in pH.
– Aspartic protease family eg. Pepsin, HIV protease,
63. Mechanisms of enzyme action
• Covalent Catalysis
– There is transient formation of covalent bonds between
the enzyme and substrate(s)
• Making the modified enzyme a reactant
– This new/hybrid species introduces an alternative
reaction pathway with lower activation energy
– On completion of the reaction the enzyme is
regenerated back to its unmodified state
66. Mechanisms of enzyme action
–This is a common mechanism among
enzymes catalyzing group transfer reactions
• Ser, Cys and occasionally His are generally the
residues that participate in covalent catalysis
• Chymotrypsin and the Ser-His-Asp network
that relays protons in the hydrolysis of peptide
bond
67. Mechanism of enzyme action
• The active site is not a dormant/passive pocket for
binding substrate.
• It is a complex molecular machine where various
chemical mechanisms occur to facilitate the conversion
of substrate to product.
• The active site provides catalytic groups (atoms, ions
and chemical interactions) that enhance the probability
that the transition state is formed
• Conserved catalytic residues among enzymes catalyzing
similar reactions
68. Catalytic efficiency
• Most enzyme catalysed reactions are highly efficient,
proceeding from 10³ to 10⁸ times faster than the
uncatalyzed reactions.
– Most reactions that occur readily in living cells would occur
too slowly to support life in the absence of these biocatalysts
– Examples: peroxidase provides alternate pathway with low
activation energy causing an increase in rate of reaction
almost 10¹ᴼ
– Carbonic anhydrase increase rate of CO₂ hydration by 10⁷
• The number of molecules of substrate converted to
product per enzyme molecule per second is called the
turnover number.
69. Enzyme specificity
• Enzymes show specificity in the reactions they
catalyze. (Broad and absolute)
– Hexokinase has broad specificity, it acts on hexose sugars
(Glucose, Mannose, Fructose)
– Glucokinase is specific for only Glucose
• A few enzymes exhibit absolute specificity; that is,
they will catalyze only one particular reaction.
• Other enzymes will be specific for a particular type
of chemical bond or functional group.
– In general, there are four distinct types of specificity
70. Types of enzyme specificity
• Absolute specificity - the enzyme will catalyze only
one reaction.
• Group specificity - the enzyme will act only on
molecules that have specific functional groups,
such as amino, phosphate and methyl groups.
• Linkage specificity - the enzyme will act on a
particular type of chemical bond regardless of the
rest of the molecular structure.
• Stereochemical specificity - the enzyme will act on
a particular steric or optical isomer.
73. Potential for regulation
• The activities of many enzymes can be regulated. i.e
enzymes can be activated and inhibited, so that the
rate of product formation corresponds to the needs
of the cell.
• Regulation of enzyme activity is a common
phenomena to all living organisms
74. Potential for regulation
• Enzyme activities can be regulated in many
ways
–Regulation of enzyme activity
• Allosteric regulation
• Association and disassociation
• Proteolytic cleavage
• Covalent modification
– Regulation of enzyme amount
• Regulation of protein synthesis (gene expression)
• Protein degradation
75. Factors affecting enzyme activity
• Temperature: The rate of an enzyme-catalyzed reaction
often increases with increasing temperature up to optimum
point, then it decreases because enzymes are thermo-
labile.
– Temperature disrupts hydrogen bonds,
– Temperature alters protein shape (denature).
– The enzyme becomes denatured; the shape of its active
site is altered and substrate cannot fit.
– The rate of reaction decreases
76.
77. • pH: A change in pH can alter the rates of enzyme-catalysed
reactions since hydrogen ion concentration disrupts bonds
between amino acids.
– Many enzyme exhibit a bell-shaped curve when enzyme
activity is plotted against pH.
78. Changes in pH influence:
– The ionization state of the substrate or the
enzyme-binding site for substrate
– The ionization state of the catalytic site (active
site) of the enzyme
– The ionization state of the protein molecule so
that their conformation and catalytic activity
changes
79. • Substrate Concentration: Increased substrate
concentration increases reaction rate until all
enzyme molecules are engaged, then reaction rate
plateaus
• Enzyme Concentration: Increased enzyme
concentration increases reaction rate until all
substrate is used up, then reaction rate decreases.
• Product inhibition: Sometimes when the product
accumulates, it can inhibit some enzymes. This type
of control limits the rate of formation of the
product when the product is underused.
80. Isoenzymes
• Also referred to as isozymes
• Are different proteins that catalyze the same
reactions – distinct enzyme forms
• Products of different genes
81. Isoenzymes
• They exhibit some differences in
properties
–Sensitivity to some regulatory factors
–Substrate affinity
• Provide the same enzymatic function but
adapted to different tissue or cellular
location, circumstances
82. Enzyme Nomenclature
• Each enzyme is assigned two names
• Recommended name: short and easy for general use
• Systematic name: more complete and used when an
enzyme has to be identified without ambiguity
• Except for some of the originally studied
enzymes such as pepsin, rennin, and trypsin,
most enzyme names end in "ase".
83. 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.
E.g, Pepsin, Trypsin etc. Which give no
hint of the associated substrate or
enzymatic rxn.
84. Enzyme Nomenclature
• The International Union of Biochemistry and
Molecular Biology (IUBMB) initiated
standards of enzyme nomenclature which
recommend that enzyme names indicate
both the substrate acted upon and the type
of reaction catalyzed
• Enzymes can also be identified through a
numerical classification scheme whereby
each enzyme has an Enzyme commission
(EC) number
85. Enzyme Nomenclature
• Recommended name:
–Most commonly used names.
–They have the suffix “-ase” attached to the
substrate of the reaction (eg, glucosidase, urease,
sucrase) or to a description of the action the
enzyme performs (eg, lactate dehydrogenase)
86. Enzyme Nomenclature
• Systematic name:
–Specifies the substrate(s) and the functional
groups acted upon
–The type of reaction catalyzed
–The suffix “-ase” is attached to a fairly
complete description of the chemical rxn
catalyzed
• Eg. Urea amidohydrolase (Urease)
–In systematic naming enzymes are divided
into 6 major classes
87.
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95. Enzyme proteins and Cofactors
• Among the enzymes, there is considerable
diversity of structure
• Many enzymes are simple protein, meaning
that the protein itself is the true catalyst.
96. Enzyme proteins and Cofactors
• Many enzymes catalyze reactions of their
substrates only in the presence of specific
non-protein molecules or metal ions.
–These non protein molecules and metal ions
required for enzyme activity are called cofactors.
–Cofactors are divided into 3 groups:
• Metal ions, coenzymes and prosthetic groups
• Inorganic [metallic ions] and organic cofactors
[coenzymes ( fv and prosthetic groups)]
97. Metal ion Cofactors
• These function primarily by forming complexes
with the enzyme itself or with other non protein
groups required by the enzyme for catalytic
activity.
98. Metal ion Cofactors
• In some cases the metal ions appear to be only
loosely associated with the active enzyme and can
be easily removed from the enzyme while in other
cases they form an integral part of the enzyme
structure
– Eg. Carbonic anhydrase requires one Zn²⁺ per molecule
of enzyme for activity.
• Other metal ions include Mg²⁺, Fe²⁺, Mn²⁺ etc.
100. Coenzymes and Vitamins
• A coenzyme is a small organic molecule that binds
reversibly to an enzyme and is required for activity
of the enzyme
• Coenzymes act as transient carriers of specific
functional groups. Most are derived from B vitamins
