ENZYMOLOGY (KIUT Year 1)-2.pptx

ENZYMOLOGY
BY
Dr Diana O. Odey

COURSE CONTENT
 Introduction
 Properties of enzymes
 Classification of enzymes
 Enzyme nomenclature
 Coenzymes and cofactors
 Factors affecting enzyme activity
 Mechanisms of enzyme activity
 Enzyme kinetics

COURSE CONTENT CONT’D
 Enzyme regulation
 Diagnostic and therapeutic importance of enzymes
 Isoenzymes; types and clinical significance

Introduction
 Catalysis is a fundamental condition for life in biological systems
 For an organism to survive, it must be able to catalyze chemical
reactions efficiently and selectively
 Enzymes are central to every biological process
 They act in an organized and step-wise manner to catalyze several
biological reactions that break down nutrient molecules, conserve and
transform chemical energy, and make biological macromolecules from
simple precursors.

Introduction cont’d
 Enzymes are biological catalysts that speed up the rate of biological
reactions, without altering the equilibrium of the reaction.
 Biological catalysis was first recognized and described in the late
1700s in studies that were centred on the digestion of meat by
secretions from the stomach.
 The name “enzyme” originated from the Greek word “enzymos” which
means “leavened”

Properties of enzymes
 All enzymes are proteins EXCEPT for a small group of catalytic RNA
molecules known as ribozymes.
 The catalytic activity of enzymes largely depends on the integrity of
their protein structure. This means that if an enzyme is denatured (or
broken into its constituent subunits or amino acids), the catalytic
activity of the enzyme is lost.
 Enzymes have molecular weights ranging from 12,000 to 1,000, 000
Daltons.

Properties of enzymes cont’d
 Some enzymes do not require any additional chemical groups for their
catalytic activity other than their constituent amino acid residues
 However, some other enzymes require an additional chemical
component called a cofactor (e.g. mostly inorganic ions like Fe2+,
Mg2+, Mn2+, or Zn2+ OR a complex organic or metalloorganic
molecule called a coenzyme).
 Note: Coenzymes are mostly derived from vitamins or other organic
nutrients required in small amounts from the diet.

 Some enzymes need both a coenzyme and one or more metal ions for
activity
 A coenzyme or metal ion that is very tightly bound to the enzyme
protein is called a prosthetic group.
 An enzyme with complete catalytic activity together with its bound
coenzyme and/or cofactor is called a holoenzyme.
 The protein part of a holoenzyme is called the apoenzyme or
apoprotein.

 Enzyme proteins can undergo covalent modification through certain
reactions (e.g. phosphorylation reactions). Many of these
alterations/modifications are involved in the regulation of enzyme
activity.
 Enzymes are neither consumed nor permanently altered in the course
of a reaction they catalyze.
 Enzymes are extremely selective/specific. Enzymes are specific for the
type of reaction catalyzed and also for a single substrate or small group
of closely related substrates.

 Enzymes are also stereospecific catalysts. This means that they
typically catalyze reactions of only one stereoisomer of a given
compound. For instance; D-sugars but not L-sugars.

CLASSIFICATION OF ENZYMES
 Enzymes can be classified based on the following criteria:
 Reaction type
 Site of action
 Substrate on which they act

CLASSIFICATION OF ENZYMES CONT’D
 Enzymes can be classified based on reaction type into six (6);
 Oxidoreductases – Responsible for the transfer of Hydride ions (H-) or hydrogen
atoms or the addition of oxygen e.g. succinate dehydrogenase enzyme (from the
TCA cycle)
 Transferases – Responsible for functional group (e.g. –NH3 ,) transfer reactions e.g.
 Hydrolases – Responsible for hydrolytic cleavage reactions e.g. lipase, protease, etc.
 Lyases – Responsible for non-hydrolytic cleavage of C-C, C-O, C-N or other bonds
by elimination, leaving double bonds or rings, or addition of groups to double
bonds e.g. aldolase (from the glycolysis pathway)
 Isomerases – Responsible for transfer of groups within molecules to yield isomeric
forms e.g. dextrose isomerase which converts dextrose to fructose.

 Ligases – responsible for catalysing the formation of C—C, C—S, C—O,
and C—N bonds or other bonds by condensation reactions coupled to
cleavage of ATP or similar cofactor e.g. DNA ligase

 Enzymes can also be classified based on the site of action into four (4);
 Endoenzymes – They are also known as intracellular enzymes. They act only inside
the cell.
Examples – Isomerases, phosphorylases
 Exoenzymes – They are also known as extracellular enzymes. They are secreted
outside the cell. They are usually digestive in their function. They hydrolyse very
complex molecules into smaller compounds.
 Examples – proteases, lipases.

 Constitutive enzymes – These are enzymes produced in the absence of
substrate
They are produced at a constant rate and in constant amount in an
organism’s metabolic state
 Constitutive enzymes are part of the basic and permanent enzymatic action
of the cell
 Example; the enzymes of the glycolytic pathway
 Induced enzymes – These enzymes are present in trace (very small)
amounts, but their concentration increases in the presence of some
substrates on which they act

 Some microorganisms produce induced enzymes in response to the
presence of certain substrates in the environment
 Some chemical compounds are also capable of triggering the
production of induced enzymes.
 Example; ethanol, barbiturates strongly induce hepatic microsomal
enzymes such as cytochrome p450 group of enzymes.

 Enzymes can be classified based on the substrate on which they act;
 Amylolytic enzymes or carbohydrases - These enzymes act only on
carbohydrates
Examples; ptyalin, maltase, etc.
 Proteolytic enzymes or proteases – These enzymes act on proteins and
hydrolyse them
Examples; trypsin, pepsin, renin, etc.
 Lipolytic enzymes – These enzymes act on lipids.
 Examples; lipase, pancreatin, etc.

ENZYME NOMENCLATURE
 There are many methods for naming enzymes;
 Old/trivial name e.g. pepsin, trypsin
 The name of the substrate with the addition of the suffix “–ase” e.g. lactase
acting on lactose
 Two-word naming, one word for the substrate and the other word for the
type of reaction e.g. succinate dehydrogenase
 IUBMB (International Union of Biochemistry and Molecular Biology)
enzyme nomenclature

IUBMB Enzyme Nomenclature
 Each enzyme is described by a sequence of four (4) numbers preceded
by the abbreviation “EC”
 “EC” stands for “Enzyme Commission” and the sequence of four
numbers that follows after the EC is called the “EC number”
 When enzymes are classified based on the IUBMB enzyme
nomenclature, the enzymes are assigned an EC number, in the form of
four digits separated by dots.

IUBMB enzyme nomenclature cont’d
 The first digit denotes the class (reaction type) of the enzyme
 The second digit denotes the functional group (subclass) upon which
the enzyme acts
 The third digit denotes the coenzyme (sub-subclass)
 The fourth digit denotes the substrate (the serial number identifies the
enzyme within the sub-class)

IUBMB enzyme nomenclature cont’d

COENZYMES AND COFACTORS CONT’D
 Cofactors are non-protein
molecules that assist enzymes
during the catalysis of reactions
 Cofactors can either be metal
ions or small organic molecules
(coenzymes)

 The complete catalytically
active enzyme is called a
holoenzyme
 The protein part of the enzyme
Without the cofactor attached is
called the apoenzyme

 Unlike prosthetic groups which are tightly bound cofactors, cofactors
which are loosely bound are transient (temporary) and dissociate easily
from either the enzyme or substrate depending on where they were
bound.
 Prosthetic groups are tightly integrated into the enzyme structure by
covalent or non-covalent forces.
 Prosthetic groups can either be organic e.g. pyridoxal phosphate (from
vitamin pyridoxine or vitamin B6), flavin mononucleotide (abbreviated
as FMN from riboflavin vitamin B2), etc.

 Prosthetic groups can also
be inorganic (e.g. metal ions)
 Metal ions are the most
common prosthetic groups
e.g. Co, Cu, Mn, Zn, Mg, Fe

 Enzymes that contain tightly bound metal ions are called
metalloenzymes
 Enzymes that require metal ions as loosely bound cofactors are
called metal-activated enzymes
 The role of metal ions as cofactors include;
Helps with the binding and orientation of the substrate
Aids the formation of covalent bonds with reaction intermediates
 Makes the substrate more electrophilic/nucleophilic (i.e. makes the
substrate more easy to react with)

A table showing some examples of metalloenzymes

 Metal activated enzymes/Ion activated enzymes in a few reactions require the
presence of certain ions to increase the reaction rate of the reaction
The ions may combine with the substrate or the enzyme
 The ion binding makes the formation of the enzyme-substrate complex much
easier by adjusting the charge distribution between the substrate and the
enzyme, as well as the end shape of the enzyme-substrate complex
 For example; amylase catalyses the breakdown of starch to maltose
molecules. However, amylase can only function properly in the presence of
chloride ions

 Coenzymes serve as “recyclable shuttle” or group transfer agents that
transport many substrates or groups from where they are generated to
where they are used
 The water-soluble B vitamins form a significant portion of most
coenzymes
 Chemical groups that can be transferred by coenzymes include;
 hydrogen atoms, or hydride ions
 methyl groups
Acyl groups, etc.

 There are two (2) major classes of coenzymes;
Class-1 Coenzymes – they transport hydrogen and electrons
 Class-2 Coenzymes – they transport other groups or moieties other than
hydrogen and electrons

 A table showing Class 1 Coenzymes

 A table showing Class 2 Coenzymes

FACTORS AFFECTING ENZYME ACTIVITY
 A basic enzymatic reaction is represented in the equation below:
 E represents the enzyme catalysing the reaction
 S represents the substrate of the reaction (i.e. the substance being
changed
 P represents the product of the reaction

CONT’D
 The image below represents an overview of general enzyme catalysis

CONT’D
 There are numerous factors that can affect enzyme activity (reaction
rate). Some of them include;

CONT’D
Effect of substrate concentration
 At lower concentration of substrate, the active sites of the enzyme
molecules are not fully occupied because the concentration (amount) of
the substrate is low
 However, at higher concentration of the substrate, there are more
collisions between the active site of the enzyme molecules and the
substrate
 Therefore, enzyme activity and rate of reaction increases.

CONT’D
 As the substrate concentration increases, the velocity (speed) of the
reaction increases.
 When the substrate concentration is almost proportional to the
reaction velocity then the reaction is called a first order reaction with
respect to substrate concentration.
 When the substrate concentration is much higher, the reaction is
called a zero order reaction because the rate of the reaction is
independent of the substrate concentration.

CONT’D
 The chart below is plot of enzyme activity(enzyme rate of reaction)
against substrate concentration.

CONT’D
 [S] – stands for substrate concentration
 Km - stands for Michaelis constant (Km is the substrate concentration at
which enzyme achieves half of the reaction’s maximum velocity)
 Vmax - stands for the maximum reaction velocity

CONT’D
Significance of Km
 Km is the substrate concentration at which the initial velocity of the
reaction is half of the maximum velocity (Vmax /2) attainable at a
particular enzyme concentration.
 It is specific and constant for a particular enzyme under defined
conditions of time, temperature and pH.
 Km can also be considered the approximate measure of the affinity of
an enzyme for its substrate. The lower the Km , the higher the affinity.

CONT’D
Enzyme concentration
 The enzyme concentration increases enzyme activity and rate of
reaction
 However, when the enzyme concentration exceeds the concentration
required for the reaction, the rate of reaction remains constant at that
point.
 Therefore, reaction rate increases as the enzyme concentration
increases until it levels off.

CONT’D
Effect of product concentration
 When there is accumulation of product, the enzyme activity begins to
decline. This is called product or feedback inhibition.
 Under certain suitable conditions, the reverse reaction may be
favoured, forming the substrate.

CONT’D
Effect of temperature
 Raising the temperature of the reaction increases the kinetic energy of
the reacting molecules.
 The increase in kinetic energy of the reacting molecules also increases
their motion and therefore the frequency with which the molecules
collide.
 Therefore, the combination of more frequent and more highly
energetic and productive collisions between reacting molecules
increases the rate of the reaction.

CONT’D
 The temperature coefficient is the factor by which the rate of a
biologic process increases for a 10 ℃ increase in temperature
 For the temperatures that do not affect the stability of an enzyme, the
rates of most biological processes typically double for a 10 ℃ rise in
temperature.
 A ten degree centigrade rise in temperature will increase the activity of
most enzymes by 50 to 100%.

CONT’D

CONT’D
 The optimal of enzymes in higher organisms rarely exceeds 50 ℃
 There are some enzymes that lose their catalytic activity/ability when
frozen
 Enzymes from thermophilic bacteria found in hot springs are active
even at 100 ℃.
 Changes in the rates of enzyme-catalysed reactions that accompany a
rise or fall in body temperature constitute a prominent survival feature
for “cold-blooded” life forms such as lizards or fish, whose body
temperatures are dictated by their external environment.

CONT’D
Effect of hydrogen ion concentration (pH)
 The rate of enzyme-catalysed reactions depend significantly on the
hydrogen ion concentration
 Most intracellular enzymes exhibit optimal activity at pH values
between 5 and 9
 When the activity is plotted against pH, a bell-shaped curve is usually
obtained

CONT’D
 The pH optimum is the pH value at which enzyme activity is at its
maximum. It is often close to the pH value of the cells (i.e. pH 7)
 However, there are some exceptions to this.
 The proteinase pepsin, which is active in the gastric lumen, has a pH
optimum of 2, while alkaline phosphatase is active at pH values higher
than 9.

CONT’D
Effect of activators and coenzymes
 The activity of certain enzymes is greatly dependent on metal ion
activators and coenzymes
 Vitamins act as coenzymes in a variety of reactions.
Effect of modulators and inhibitors
 The activity of enzymes is reduced in the presence of an inhibitor.
 The activity of an enzyme may also be increased in the presence of a
positive modifier/modulator.

CONT’D
Effect of time
 The longer an enzyme is incubated with its substrate, the greater the
amount of product that will be formed.
 Enzyme catalysed reactions are completed with optimum time.
 Variations of time affect the rates of enzyme catalysed reactions
 However, it important to note that the rate of formation of product is
not a simply a function of the time of incubation.
 Also note that all proteins suffer denaturation, and hence loss of
catalytic activity with time.

MECHANISM OF ENZYME ACTIVITY
 In order for enzymes to carry out their functions of speeding up the
rate of biochemical reactions, they make use of some “catalytic
strategies”.
 The energy required by reactants to undergo a reaction is called the
Gibbs free energy of activation (activation energy).
 Enzymes work towards lowering the activation energy of the reaction
in order to speed up catalysis.
 The highest point of activation that reactants reach in the course of a
reaction is called the transition state.

CONT’D
 Note: Reactants in the low energy state (at the beginning of a reaction) are
usually quite stable and will usually require a substantial amount of energy
(activation energy) to reach its transition state (the peak of the reaction).
 EA = activation energy
 ΔG < 0 = Standard
Gibbs Free energy change
(net change in energy level
between the reactants and
products)

CONT’D
 As enzymes lower the activation energy of the reaction, they equally
make the highly unstable and transient transition state of the reactants
more stable, proceeding to the formation of the product faster.
The formation of the enzyme-substrate (ES) complex
 For enzyme catalysis to occur, the substrate must combine with the
enzyme at the enzyme’s active site to form an ES complex, which
eventually leads to the formation of a product.
 Some theories have been put forward to explain how the ES complex
is formed. They are;

CONT’D
 Lock and key model or Fischer’s template theory
 Induced fit theory or Kosherland’s model
 Substrate strain theory
Lock and key model or Fischer’s template theory
 The lock and key theory was proposed by Emil Fischer, a German
biochemist
 He proposed the theory to explain enzyme-catalysed reactions
 His model suggests that an enzyme has a rigid (inflexible) structure.

CONT’D
 The model also suggests that the substrate on binding to the enzyme
fits into the enzyme’s active site just the way a key fits into its lock or a
hand into the proper glove
 In other words, the model describes the active site of the enzyme as
fixed (rigid), with a pre-determined shape, where only a specific
substrate can bind.
 This model is limited because it does not give room to explain the
flexible nature of enzymes. The model also fails to explain many facts
about enzymatic reactions especially concepts like the effect of
allosteric modulators.

CONT’D
Induced fit theory or Kosherland’s model
 This model was proposed by Kosherland in 1958.
 It is a more acceptable and realistic theory that explains the formation
of the ES complex
 Unlike the Fisher’s model, the induced fit theory does not represent
the active site of an enzyme as rigid or pre-shaped
 Rather, this model explains that the interaction of the substrate with
the enzyme induces a conformational change to the active site of the
enzyme, that allows the formation of a strong substrate binding site.

CONT’D
 The induced fit model also suggests that the appropriate amino acids
of the enzyme are properly repositioned to form the active site and
promote catalysis.
 Unlike the Fisher’s model, the induced fit model has sufficient
experimental evidence from X-ray diffraction studies to back up its
claims.
 The induced fit model also supports the concept of allosteric
modulators and competitive inhibition of enzymes

CONT’D
Substrate strain theory
 This theory suggests that the substrate becomes strained after
induced conformational (structural) changes have occurred in the
enzyme.
 The model also suggests that there is a possibility that when a
substrate binds to the pre-formed active site, the enzyme could induce
a strain to the substrate.
 The strained substrate leads to the formation of product.
 A combination of the induced fit model and the substrate strain theory
is considered to be what happens in enzymatic action.

CONT’D
The Lock and key model
Illustration

CONT’D
Mechanism of enzyme catalysis
 Enzyme catalysis is believed to occur based on four processes;
 Acid-base catalysis
Substrate strain
 Covalent catalysis
 Proximity catalysis

CONT’D
 Acid-base Catalysis
 This refers to a process where enzymes act as either an acid or base as a
strategy to promote catalysis
 Its important to remember that, acids and bases function as proton donors
and acceptors
 Therefore, enzymes make proton transfer between intermediates faster
since they can act as proton donors and acceptors, thereby enhancing
catalysis and speeding up the rate of the reaction.
 For instance, histidine an amino acid found in some enzymes can be
protonated and act as an acid and its corresponding conjugate as a base.

CONT’D
 Substrate strain
 When a strain is being induced on a substrate during enzyme-substrate
complex formation, the energy level of the substrate is raised, leading to a
transition state.
 For instance the action of the enzyme lysozyme (is an enzyme of the innate
immune system with strong antimicrobial activities; it cleaves the
peptidoglycan component of bacterial cell walls leading to the death of the
bacterium) is attributed to a combination of substrate strain and acid-base
catalysis.

CONT’D
 Covalent catalysis
 Covalent catalysis involves negatively charged (nucleophilic) or positively
charged (electrophilic) groups present at the active site of the enzyme.
 These groups are capable of attacking the substrate, resulting in the
covalent binding of the substrate to the enzyme.
 For instance, in serine proteases like trypsin and chymotrypsin, the covalent
catalysis occurs alongside acid base catalysis to promote the reaction
catalysis.

CONT’D
 Proximity Catalysis
 For catalysis to occur, the reactants should come in close proximity to the
enzyme.
 The higher the concentration of the substrate molecules, the greater the
rate of the reaction will be.
 As the enzyme binds to the molecules of the substrate at the enzyme’s
active site, there will be a corresponding several fold increase in catalysis.
Note: In actual enzyme catalysis, more than one of the processes listed above
is employed simultaneously.

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