Enzymes
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Enzymes – Classification
 Enzymes are proteins that act as catalysts. They increase the rate at which chemical
reactions occur without being consumed or permanently altered themselves
 Enzymes are classified according to the reactions that they catalyze:
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Enzymes – Classification
1. Oxidoreductases: they catalyze the transfer of electrons from one molecule, the
reductant (electron donor) to another, the oxidant (electron acceptor). They can
be oxidases or dehydrogenases
• Oxidases are involved when oxygen acts as an acceptor of hydrogen or electrons
• Dehydrogenases oxidize a substrate by transferring hydrogen to an acceptor
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Enzymes – Classification
2. Transferases: they transfer a chemical group
from one molecule to another.
•
•
•
•
•
•
Acyltransferase
Aminotransferase
Glycosyltransferase
Kinase
Methyltransferase
Nucleotidyltransferase…
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Enzymes – Classification
3. Hydrolases: They split water and add it to molecules. They catalyze the hydrolytic
cleavage of carbon-oxygen, carbon-nitrogen, and carbon-carbon bonds.
• Phosphatase
• Peptidase
• Lipase
• Hydrolase
• Amylase
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Enzymes – Classification
4. Lyases: they break double bonds trough the addition of water, ammonia, or carbon
dioxide. They can also form double bonds by the removal of these groups from the
substrate
•
•
•
•
•
Decarboxylase
Synthase
Aldolase
Cyclase
Endonuclease
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Enzymes – Classification
5. Isomerases: they catalyze reactions that change or alter small parts of the
substrate inducing intramolecular rearrangements
• Epimerase
• Mutase
• Racemase
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Enzymes – Classification
6. Ligases: also called Synthetase, they
catalyze the joining of two molecules,
using energy from ATP  ADP
• amino acid–RNA ligase: catalyzes
the formation of a carbon-oxygen
(C―O) bond between an amino acid
& transfer RNA
• amide synthetases & peptide synthetases: catalyzes the formation of Carbon–
nitrogen (C―N) bonds
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Enzymes – Nomenclature
 Usually ends in ase
 Identifies the reacting substrate; for example:
• urease catalyzes the reaction of urea
• lactase catalyzes the reaction of lactose
 Describes the function of the enzyme
• Dehydrogenase
• oxidase
• decarboxylase
 Can be common name, with no direct relationship to substrate or reaction type, particularly
for the digestive enzymes
• Pepsin
• Chymotrypsin
• Trypsin
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Enzymes – Energy of reactions
 An enzyme speeds a reaction by lowering the activation energy, changing the
reaction pathway
 This provides a lower energy route for conversion of substrate to product
 Every chemical reaction is characterized by an equilibrium constant Keq, which is a
reflection of the difference in energy between reactants aA, and products, bB
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Enzymes – Activation energy
 As substrates are transformed into products during a chemical reaction, they go
through an intermediate transition state
 The chemical reaction is at its highest
energy at the transition state
 The difference between the energy level
of the substrate and the energy level of the transition state is called the activation
energy
 Activation energy is the energy needed to overcome the energy barrier of breaking
and reforming bonds for a reaction to proceed.
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Enzymes – Activation energy
 During a chemical reaction, substrates (A + BC) reach a transition state (A—B—C)
before they are transformed into products (AB + C)
 Compared to an uncatalyzed reaction (left), enzymes lower the activation energy
by stabilizing the transition state to an energetically favorable conformation (right)
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Catalyzed & uncatalyzed reactions
 The rates of uncatalyzed reactions increase as the substrate concentration increases
 The rates of catalyzed reactions are limited by enzyme availability & show two stages:
• The 1st stage is the formation of an enzyme-substrate complex
• The 2nd stage is a slow conversion to product
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Reactions & substrate concentration
 Increasing substrate concentration increases the
frequency with which the enzyme & substrate collide
 As a result enzyme-substrate complexes form
more quickly and the rate of reaction increases
 However, there is a limit as eventually there all the enzyme active sites are already
occupied with substrate  the enzyme active sites become saturated
 Any further increase in substrate concentration has no further effect on the
reaction rate
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Reactions & enzyme concentration
 Increased enzyme concentration  increase the
formation of enzyme-substrate complexes  accelerate
the reaction’s rate
 BUT, if more enzyme molecules present than substrate
 some enzymes won't have any substrate to bind to
 Increased enzyme concentration  no further effect on the reaction’s rate
 If there is excess substrate  1st part of the curve can be approx. to a straight line
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Enzyme-substrate complex
The reversible enzyme-catalyzed reaction steps:
• Formation of a temporary enzyme-substrate complex
(E-S) when an enzyme comes into contact with its
substrate  conformational change when the
substrate enters the active site (E-S is the transition
state)
• Formation of the enzyme-product complex (E-P) and when products are released,
the enzyme is ready for another substrate
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Enzyme-substrate complex details
 The part of the enzyme combining with the substrate is the active site
 Active sites characteristics include:
• Pockets or clefts in the surface of the enzyme
• R groups at active site are called catalytic groups
• Shape of active site is complimentary to the shape of the substrate
• The enzyme attracts & holds the substrate using weak non-covalent interactions
• Conformation of the active site determines the specificity of the enzyme
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The lock & key enzyme model
In this model, the enzyme is assumed to be the lock and the substrate is
the key and that both have fixed confirmation that lead to an easy fit
• The enzyme and substrate have fixed conformations that lead to an easy fit
• This model fails to take into account proteins conformational changes to
accommodate a substrate molecule
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The induced-fit enzyme model
In this model, it is postulated that the enzyme’s active site is a flexible, not rigid,
pocket
• Upon exposure the shapes of the enzyme, active site, and substrate adjust to
maximize the fit and form the E-S complex  improving the catalytic reaction
• There is s greater range of substrate
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Enzyme-substrate complex specificity
For an enzyme & substrate to react,
surfaces of each must be complementary
Enzyme specificity: the ability of an
enzyme to bind only one, or a very few,
substrates  catalyzing only a single
reaction
Example: Urease is VERY Specific or has a
HIGH DEGREE of Specificity
Classes of enzyme specificity
1.
Absolute – one enzyme acts only on one substrate (Ex. Urease decomposes only
urea; arginase splits only arginine)
2.
Group – one enzyme can catalyze one type of reaction for similar substrates (Ex.
hexokinase adds a phosphate group to different hexoses)
3.
Relative/linkage - one enzyme acts on different substrates which have the same
bond type (Ex. pepsin splits different proteins)
4.
Stereochemical – some enzymes can only catalyze the transformation of substrates
which are in certain geometrical configuration (Ex. enantiomers)
Cofactors & Coenzymes
 Active enzyme / Holoenzyme:
• Polypeptide portion of enzyme (apoenzyme)
• Nonprotein prosthetic group (cofactor)
 Cofactors are bound to the enzyme for it to
maintain the correct configuration of the active site
• Organometallic compounds
• Metal ions
• Organic compounds
Cofactors & Coenzymes
 A large number of enzymes require an additional non-protein component to carry
out its catalytic functions called as cofactors
 A coenzyme or metal ion is covalently bound to the enzyme protein is called
prosthetic group
 Cofactors-two types:
• Inorganic ions such as Fe2+, Mg2+, Mn2+, Zn2+
• A complex organic molecule called coenzyme
 Some enzymes require both a coenzyme & one or more metal ions for the activity
 Coenzymes function as transient carriers of specific functional groups
Coenzymes
 They are essential for the biological activity of the enzyme and act as group transfer
reagents (Hydrogen, electrons, or other groups can be transferred)
 They are low molecular weight organic substances, bound to the enzyme by weak
interactions /hydrogen bonds, without which the enzyme can’t exhibit any reaction
Coenzymes classification
 Metabolite coenzymes – synthetized from common metabolites and include
nucleoside triphosphates
 Vitamin-derived coenzymes – derivatives of vitamins
• vitamins are required for coenzyme synthesis &
must be obtained from diet
• Most vitamins must be enzymatically transformed to the coenzyme
Water-soluble vitamins & their coenzymes
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Environmental effects
 The environment surrounding an enzyme can have a direct effect on
enzyme function
 Each enzyme exhibits peak activity at narrow
pH range – optimum pH
 Enzymes contain R groups of aa with proper charges
at optimum pH
 Extreme pH changes will denature the enzyme  disrupt the tertiary
structure  destroy the catalytic activity
• Pepsin (stomach)
• Trypsin (small intestine) have different optimum pH
Temperature (T°) effects
 An enzyme has an optimum T° associated with
maximal function
 The rate of an uncatalyzed reaction will increase
proportionally with T° increase
 Optimum T° is usually close to the T° at which the
enzyme typically exists (37oC for humans)
 Excessive heat can denature a enzyme making it
completely nonfunctional
Measuring enzyme’s kinetics
 What is kinetics? – it is an approach to learn a reaction’s mechanism by studying
factors that influence its rate
 How does this apply to enzymes? – The mystery with enzymes is to know what
the enzyme is doing, when it will function, what is it doing, and how factors affect
its rate
 More… – Does the enzyme reacts with the substrate directly? What concentration
is needed? How should an enzyme be essayed? How does an inhibitor affect
enzymatic activity? Kinetics ginve these answers and a whole lot more
Substrate concentration & enzyme activity
At low [sub], there is a steep increase in the rate of reaction with increasing [sub]. The
catalytic site of the enzyme is empty, waiting for substrate to bind, and the rate at
which product can be formed is limited by the [sub] which is available
Substrate concentration & enzyme activity
As the [sub] increases, the enzyme becomes saturated with substrate. When the
catalytic site is empty, more substrate binds and undergoes reaction. The rate of
formation of product depends on the activity of the enzyme itself, and adding more
substrate will not affect the rate of the reaction to any significant effect
Substrate concentration & enzyme activity
 Maximum rate of reaction (Vmax) - rate of reaction
when the enzyme is saturated with substrate
 The relationship between Vmax and [sub] depends
on the affinity of the enzyme for its substrate.
 This is usually expressed as the Km (Michaelis-Menten constant) of the
enzyme, an inverse measure of affinity
 Km - concentration of substrate which permits the enzyme to achieve ½ Vmax
 An enzyme with a high Km has a low affinity for its substrate, and requires a
greater concentration of substrate to achieve Vmax
How to determine Km & Vmax
 Km and Vmax are determined by incubating the enzyme with varying concentrations
of substrate; the results can be plotted as a graph of rate of reaction (v) against
concentration of substrate [S] (a hyperbolic curve)
 The relationship is defined by the Michaelis-Menten equation:
Vo = Vmax [S] / (Km + [S])
K , K , and K are specific
rate constants
k1
k2
E+S
ES
E+P
k-1
1
-1
2
If assume [S] >>>>>[E] then ES will be fairly constant. The rate of rxn is determined by [ES]
•Michaelis-Menten constant
Km = (k2 + k-1) / k1
• When V0=1/2Vmax, Km=[S]
The importance of Km & Vmax
 The Km permits to predict if the rate of formation of product will be
affected by the availability of substrate
 Km reflects affinity of enzyme for its substrate
 Low Km  enzyme normally saturated with substrate, will act at a
constant rate, regardless of variations in the [sub]
The importance of Km & Vmax
 High Km  enzyme is not saturated with substrate, and its activity will
vary as the [sub] varies  the rate of formation of product depend on
the availability of substrate
 Smaller Km  enzyme has great affinity for its substrate
Km indications - hexokinase
 In most tissues, the phosphorylation of glucose (Glc) to
G6P is catalyzed by hexokinase
 It has a low Km (0.1 mM)  high affinity for Glc 
operates at Vmax under physiological blood Glc of ~5
mM
 Activity does not change with blood Glc levels  at
fasting [Glc], hexokinase is at Vmax, but glucokinase
activity varies according to [Glc]
Glucokinase vs. Hexokinase
 Hexokinase has low Km  efficiently use low levels of Glc. But quickly saturated
 Glucokinase is found in liver and β-cells of the pancreas
 Glucokinase allows liver to respond to blood Glc levels
 Glucokinase has a high Km, so it does not become
saturated till very high levels of Glc are reached
 At low Glc levels, very little taken up by liver, so is spared for other tissues.
Regulation of enzyme activity
 One of the major ways that enzymes differ from nonbiological catalysts is in the
regulation of biological catalysts by cells
 How organisms regulate enzymatic activity?
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Enzyme produced only when the substrate is present – common in bacteria
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Allosteric enzymes
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Feedback inhibition
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Zymogens - Proteolytic enzymes are synthesized as inactive precursors, to prevent unwanted protein degradation
•
Protein modification
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Allosteric enzymes
Allosteric enzyme - enzyme that contains a region (2nd site) to which small,
regulatory molecules "effectors" may bind in addition to and separate from the
substrate binding site (1st site) and thereby affect the catalytic activity.
• Positive allosteric  speed up enzymatic activity
• Negative allosteric  slow down enzymatic activity
Feedback inhibition
 Allosteric enzymes are the basis for feedback inhibition - a product late in a series
of enzyme-catalyzed reactions serves as an inhibitor for a previous allosteric
enzyme earlier in the series (enzyme’s activity is inhibited by the end product)
 This mechanism allows cells to regulate how much of an
enzyme’s end product is produced
 Example - product F serves to inhibit the activity of
enzyme E1
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Zymogens - proenzymes
 A proenzyme - the inactive form of an enzyme
 Irreversible (Zymogen) or reversible (covalent modulation)
 Zymogenes are activated when one or more peptides are removed by proteolysis
• The zymogen proinsulin is converted to its active form, insulin, by removing a small
peptide chain
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Proenzymes of the digestive tract
 Digestive enzymes are produced as zymogens, and are then activated when deeded
 Most of them are synthetized and stored in the pancreas, and then secreted into
the small intestine, where they are activated by removal of small peptide sections
 They must be stored as zymogens otherwise they would damage the pancreas
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Reversible covalent modulation
 In protein modification a chemical group is covalently added to or removed
from the protein. This modification will activate or deactivate the enzyme
 A common example is phosphorylation of an enzyme by addition of a
phosphate group to serine, threonine, or tyrosine mediated by a kinase
 The phosphorylation is reversible,
and phosphatases typically catalyze the
of the phosphate group removal
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Inhibition of enzyme activity
 Chemicals can bind to enzymes and eliminate or drastically reduce catalytic
activity
 Classify enzyme inhibitors on the basis of reversibility and competition
• Irreversible inhibitors - bind tightly to the enzyme and thereby prevent
formation of the E-S complex
• Reversible competitive inhibitors - often structurally resemble the substrate
and bind at the normal active site
• Reversible noncompetitive inhibitors - usually bind at someplace other than
the active site. Binding is weak and thus, inhibition is reversible
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Irreversible inhibitors
Irreversible enzyme inhibitors bind very tightly to the enzyme
 Binding of the inhibitor to one of the R groups of an aa in the active site
•
This binding blocks the active site  the enzyme-substrate complex can’t form
•
The inhibitor interferes with the catalytic group of the active site eliminating
catalysis
 Irreversible inhibitors include:
•
•
•
Arsenic
Snake venom
Nerve gas
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Irreversible “Suicide” inhibitors

Special type of irreversible inhibition of enzyme activity. Also known as mechanism
based inactivation

The inhibitor makes use of the enzyme’s own reaction mechanism to inactivate it

In suicide inhibition, the structural analog binds to the active site of the enzyme
and is converted to a more effective inhibitor with the help of the

The substrate-like compound initially binds with the enzyme and the first few steps
of the reaction are catalysed

This new product irreversibly binds to the enzyme and inhibits further reactions
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Suicide inhibitors - Example
Aspirin acts as an acetylating agent where an acetyl group is covalently attached
to a serine residue in the active site of the cyclooxygenase enzyme, rendering it
inactive preventing inflammation, swelling, pain and fever
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Suicide inhibitors - Example
Clavulanic acid, which inhibits β-lactamase
clavulanic acid covalently bonds to a serine reside in the active site of the β-lactamase,
restructuring the clavulanic acid molecule, creating a much more reactive species that
attacks another amino acid in the active site, permanently inactivating it, and thus
inactivating the enzyme β-lactamase.
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Reversible competitive inhibitors
 Reversible, competitive enzyme inhibitors are also called structural analogs
• Molecules that resemble the structure and charge distribution of a natural substrate
• Resemblance permits the inhibitor to occupy the enzyme active site
• Once the inhibitor is at the active site, the enzyme activity is inhibited
 Inhibition is competitive because the inhibitor and the substrate compete for binding
to the active site
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Reversible competitive inhibitors
Km increases
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Reversible, noncompetitive inhibitors
Reversible, noncompetitive enzyme inhibitors bind to R groups of amino acids or to the
metal ion cofactors
 This binding is weak
 Enzyme activity is restored when the inhibitor dissociates from the enzymeinhibitor complex
 These inhibitors:
• Do not bind to the active site No effect on Km
• Do modify the shape of the active site once bound elsewhere in the structure
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Comparison between competitive &
noncompetitive inhibitors
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Uses of enzymes in medicine
 Diagnostic – biomarker levels altered with disease
• Heart attack: Lactate dehydrogenase, Creatine phosphate
• Acute myocardial infarction: Creatine kinase, Myoglobin, Troponin I
• Pancreatitis: Amylase, Lipase
 Analytical reagents – enzyme used to measure another substance
• Urea converted to NH3 via urease: Blood urea nitrogen measured
 Replacement therapy
• Administer genetically engineered β-glucocerebrosidase for Gaucher’s disease
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Enzymes in diagnostic use
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Isoenzymes
 Isoenzymes or isozymes - multiple forms of same enzyme (different structures) that
catalyze the same chemical reaction
 Different chemical & physical properties:
• Electrophoretic mobility
• Kinetic properties
• Amino acid sequence
• Amino acid composition
 Example: lactate dehydrogenase is a tetramer with 5 isomers (different Km and Vmax)
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Thank you
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