ENZYMES

Definition:

Enzymes are organic, thermolabile biological catalysts produced by the living organisms to
increase the rate of a chemical reaction without any permanent change in their structure or being
consumed in the process.

Chemistry:

All enzymes are proteinous (contain amino acids) except for the ribozymes that are nucleic acids
(nitrogenous bases+ sugar+ phosphate group).

Enzymes

Nucleic
Proteinous
acids

Simple Complex
protein protein
enzymes enzymes

Simple protein enzymes consist of protein only while conjugated or complex protein enzymes
are composed of protein as well as non-protein part.

Apoenzyme:

An apoenzyme is an inactive enzyme consisting of protein only. The activation of the enzyme
occurs upon binding of a non-protein organic or inorganic molecule.

Holoenzyme:

An apoenzyme together with its non-protein part is called Holoenzyme. It is complete and
catalytically active.
Cofactors:

These are the non-protein component of an enzyme that activates it by removing electrons,
protons or a functional group. These may be;

 Co-enzyme (organic, thermolabile, loosely attached to the apoenzyme through non-

covalent linkages)
Examples: NAD+, FAD+, vitamins
 Prosthetic group (inorganic, thermostable, firmly attached non-protein part)
Examples: Copper, zinc, magnesium, potassium, calcium

Substrate:

A reactant that binds to an enzyme is called substrate.

Product:

The end result of a chemical reaction between two or more reactants is called product.

Active site:

An active site is the restricted region of an enzyme formed from amino acid sequences in the
polypeptide chains to which substrate binds.

Or

The catalytic site of an enzyme is called active site.

Nomenclature of enzymes:

On the name of substrate:

Enzymes can be named using the name of substrate and the suffix – ase added to it as lactase
acting on lactose and sucrose acting on sucrose.

On the name of substrate and type of reaction:

Starting with the name of substrate and the last name indicates the type of reaction e.g. xanthine
oxidase and PBG synthase.

Reaction of enzyme with substrate:

The steps involved in reaction of enzyme with substrate to yield product/s are;

 The substrate (S) binds to the enzyme (E) at the active site to form activated intermediate
“enzyme substrate complex” (ES).
 The substrate transforms into product (P) due to enzyme action
 The EP complex cleaves to form the products (P) and the original enzyme (E)
Binding of enzyme to a substrate:
Lock and key theory:
Proposed by Fischer in 1894 Size does not matter,
shape matters

The substrate fits in this catalytic site in a similar way to lock and key. The key will only fits its
own lock.
The catalytic site of the enzyme has a shape that is complementary (fit) to the shape of the
substrate.

Induced-fit theory:

Proposed by Koshland in 1958

The catalytic site of the enzyme is not complementary to the substrate. Binding of the substrate
to the enzyme induces changes in the shape of the catalytic site making it more fit for substrate.
Mechanism of action of enzymes:

The catalytic efficiency of enzymes is explained by two perspectives:

1. Thermodynamic changes
2. Processes at the active site

Thermodynamic changes:

All chemical reactions have energy barriers between reactants and products. The difference in
transitional state and substrate is called activational barrier. Enzymes increase the rate of reaction
by decreasing the activation energy of reaction. Activation energy is the energy barrier between
reactants and products.
Processes at the active site:

Four types of processes may take place at the active site of enzyme:

 Covalent catalysis
 Acid base catalysis
 Catalysis by strain
 Catalysis by proximity

Covalent catalysis

Enzymes form covalent linkages with substrate forming transient enzyme-substrate complex
with very low activation energy. Enzyme is released unaltered after completion of reaction.
Acid base catalysis

Such reactions are mostly undertaken by Oxidoreductases and the active site usually contains
histidine which acts as both proton donor and proton acceptor.

Catalysis by strain

In this catalysis, molecules must come in bond forming distance. When enzyme binds, a region
of high substrate concentration is produced at active site. This will orient substrate molecules
especially in a position ideal for them.

Catalysis by proximity

The enzyme (lyases)-substrate binding causes reorientation of the structure of site due to in a
strain condition. Thus transitional state is required and here bond is unstable and eventually
broken. In this way bond between substrate is broken and converted into products.
Factors affecting rate of enzyme action:

A number of factors affect the action of enzymes such as;

Concentration of enzyme

The rate of enzyme action is directly proportional to the concentration of enzyme provided that
there is sufficient supply of substrate & constant conditions.

Concentration of substrate

The rate of reaction increases as the substrate concentration increases up to certain point at which
the reaction rate is maximal. At Vmax, the enzyme is completely saturated with the substrate any
increase in substrate concentration doesn't affect the reaction rate.

Concentration of co-enzyme

In the conjugated enzymes that need coenzymes, the increase in the coenzyme concentration will
increase the reaction rate.

Concentration of ion-activators

The increase in metal ion activator increases the reaction rate of enzymes that are activated by
ions e.g. salivary amylase (activated by chloride ion).

Temperature

Rate of reaction increases gradually with the rise in temperature until reach a maximum at a
certain temperature, called optimum temperature. The optimum temperature is 37-40°C in
humans.

Increase of temperature causes;

1- Increase the initial energy of substrate and thus decrease the activation energy
2- Increase of collision of molecules causes change in the distances due to which bond
forming or bond breaking is easier
After the optimum temperature, the rate of reaction decrease due to denaturation of the enzyme
(60-65°C).

pH

Each enzyme has an optimum pH at which its activity is maximal e.g. Optimum pH of pepsin is
1.5-2, pancreatic lipase = 7.5-8 and salivary amylase is 6.8.

Change of pH above or below optimum decrease rate of enzyme action due to:

 The enzyme activity depends on the ionization state of both enzyme and substrate
 Marked change in PH will cause denaturation

Time

In an enzymatic reaction, the rate of reaction is decreased by time due to:

 The decrease in substrate concentration

 The accumulation of the end products
 The change in pH than optimum

Allosteric enzymes
The enzymes having two receptor sites are called allosteric enzymes. One of the site in such
enzymes is a substrate binding site while the other is reserved for inhibitors or activators.
Enzyme inhibitors

The presence of enzyme inhibitor decreases or stops the enzyme activity. Enzyme inhibitors may
be irreversible or reversible inhibitors.

Reversible inhibition:

It is an inhibition of enzyme activity in which the inhibiting molecular entity can associate and
dissociate from the protein„s binding site.

Competitive inhibition: The competitive inhibitors of an enzyme have similar shape to that of
substrate; hence compete with it for occupying the active site.

Example: Statins for lowering lipid levels compete with HMG-CoA and inhibit the enzyme
HMG-CoA reductase

Non-competitive inhibition: Inhibitors of an enzyme have different binding site to that of the
substrate and their binding results in changing the enzyme structure that prevents substrate
attachment.

Mixed inhibition: In this type of inhibition, both E.I and E.S.I complexes are formed that are
catalytically inactive.

Un-competitive inhibition: The inhibitor does not compete with the substrate for the active site
of enzyme instead it binds to another site known as allosteric site.

Example: Tetramethylene sulfoxide inhibits liver alcohol dehydrogenase

Irreversible inhibition:

This type of inhibition involves the covalent attachment of the inhibitor to the enzyme. The
catalytic activity of enzyme is completely lost. It can only be restored only by synthesizing
molecules.
Aspirin which targets and covalently modifies a key enzyme involved in inflammation is an
irreversible inhibitor of COX.
Specificity of enzymes

Enzymes are highly specific in their action. Specificity is a characteristic property of the active
site.

Types:

Stereospecificity:

Stereoisomers are the compounds which have the same molecular formula, but differ in their 3D
configuration.

The enzymes act only on one isomer and, therefore, exhibit stereospecificity e.g.

 L-amino acid oxidase and D-amino acid oxidase act on L- and D-amino acids respectively
 Glucokinase acts on D-glucose
 Alpha amylase acts on α-glycosidic linkages

Reaction specificity:

The same substrate can undergo different types of reactions, each catalyzed by a separate
enzyme and this is referred to as reaction specificity. An amino acid can undergo transamination,
oxidative deamination, decarboxylation, racemization etc. The enzymes however, are different
for each of these reactions.
Substrate specificity:

Absolute substrate specificity:

Certain enzymes act only on one substrate e.g. glucokinase acts on glucose to give glucose 6 -
phosphate, urease cleaves urea to ammonia and carbon dioxide

Relative substrate specificity:

Some enzymes act on structurally related substances i.e. these are dependent on the specific
group or a bond present.

Bond specificity:

Most of the proteolytic enzymes are showing group (bond) specificity e.g. trypsin can hydrolyse
peptide bonds formed by carboxyl groups of arginine or lysine residues in any proteins.

Group specificity:

One enzyme can catalyse the same reaction on a group of structurally similar compounds e.g.
hexokinase can catalyse phosphorylation hexoses such as glucose, galactose and mannose.
Enzyme activation

In enzymology, activators are the molecules that increase the velocity of an enzyme-catalyzed
reaction due to reversible binding to the enzyme.

Enzyme action may be;

1- Nonessential activation
The reaction can occur in the absence of the activator as well as in its presence
2- Essential activation
The reaction will not take place in the absence of an activator such as glucokinase and
hexokinase
Classification of enzymes
A number of systems mentioned below have been evolved to name and classify the enzymes
over a period of time.

 On the basis of substrate

 On the basis of the reaction catalyzed
 On the basis of substrate acted upon and type of reaction catalyzed
 On the basis of substance being synthesized

To address the unambiguity and uniformity in the classification of enzymes, a system based on
the type of chemical reaction and the mechanism is given by International Union of
Biochemistry.

Features of IUB classification:

 Enzymes are grouped in 6 classes, each with 4-13 sub-classes
 Each enzyme has two parts; First part indicates the name of the substrate and second part
ending at the suffix –ase indicates the type of reaction catalyzed. Additional information
regarding the nature of the reaction is given in parenthesis (if needed)
 Each enzyme has a unique name and code number (EC) and has four digits. These four
digits describe;

Digit 1 Class Major class of enzyme

Digit 2 Sub-class Type of group involved
Digit 3 Sub of sub-class Substrate
Digit 4 Sub of sub-sub class Serial number of a particular enzyme
Six major classes of enzymes and their functions

Oxidoreductases

Reaction type: Redox reactions in which oxygen or hydrogen are transferred

Example of enzymes: Oxidases, Reductases, Dehydrogenases

Specific examples: Alcohol dehydrogenase, Lactate dehydrogenase, Xanthine oxidase

Transferases

Reaction type: Transfer of functional group

Example of enzymes: Transaminases, transketolases, transaldolases

Specific examples: glucokinase, hexokinase, L-arginine:glycine amidinotransferase,

guanidoacetate methyl transferase
Hydrolases

Reaction type: hydrolysis

Example of enzymes: Amylases, Lipases, Proteases, Nucleases,

Specific examples: Sucrase, amidase, ribonuclease

Lyases

Reaction type: Elimination of group to form double bonds without hydrolysis

Example of enzymes: Aldolase, Decarboxylase, Fumerase

Specific examples: Histidine decarboxylase, pyruvate decarboxylase

Isomerases

Reaction type: Transfer of a group within a molecule

Example of enzymes: Isomerase, Mutase, Epimerase

Specific examples: Xylose isomerase

Ligases

Reaction type: Bond formation coupled with ATP hydrolysis

Example of enzymes: Synthases, carboxylases

Specific examples: Pyruvate carboxylase, aminolevulinate synthase, prophobilinogen synthase

Michaelis-Menten equation
The Michaelis-Menten equation is a well-known model used in enzyme kinetics. It is a special
arrangement of a two-parameter rectangular hyperbola. The mathematical model is;

Where;
V0 = Dependent variable (velocity or the rate of enzymatic reaction)
[S] = Independent variable (substrate concentration)
Vmax = maximum velocity
Km = Michaelis-Menten constant or EC50 (Vmax/2)

Purpose of deriving Michaelis-Menten equation

During an enzymatic reaction, the substrate [S] attaches to the enzyme [E] at the active site and
results in formation of product [P]. This indicates that during the course of the reaction, the
concentration of [S] decreases with subsequent increase in the [P].
The slope of the plots (t vs. [S] or t vs. [P] or t vs. [E]) gives the rate of reaction i.e.

[S] and [E] are negative because the substrates are being used and [P] is positive because product
is being formed.
Since,
Changes in the of concentration of [S] or [P] or [E] are expressed as per unit time, hence, rate of
reaction = velocity
The plot between the [S] and velocity if parabolic and can be divided into two segments;
1- A linear increase in the velocity with [S] concentration indicates 1st order reaction
2- Plateau phase where increase in [S] concentration does not increase the velocity: zero
order reaction
The 1st order reaction can be explained using the equation
Y = mx + c
Where, Y = velocity, m = slope, x = [S] concentration, and c = intercept of the plot
In contrast to the 1st segment, the 2nd i.e. zero-order cannot be explained by the abovemtioned
equation, hence MM equation was developed.

What does Michaelis-Menten equation explains?

Michaelis-Menten equation explains;
1- The plot between velocity and substrate concentration
2- Helps to derive a mathematical relationship between V0, Vmax and Km
3- Both 1st and 0th order reaction

Derivatizing Michaelis-Menten equation

The reaction between [E] and [S] to form [ES] is reversible.
Assumption 1:
According to the Law of mass action;

Where, Kr/Kf = Kd (dissociation constant)

Hence,

Assumption 2:
Pseudo steady state hypothesis states that [ES] in an enzymatic reaction remains constant which
means;
Rate of formation of ES = Breakdown of ES
We know that;

Hence,

Taking ES as common;

Or

Where; (Michaelis-Menten constant)

So,
Developing a correlation between V0, Vmax and Km
Since,

So, product formation depends on dissociation of ES.

Hence,

Or

Initially, in a system, many of the enzyme molecules do not bind with substrate;
So, the total enzyme concentration is;

Or

The velocity becomes maximum, when all the enzyme molecules are bound to the substrate i.e. E
= 0, then;

Putting the value of ES and determining the Vmax;

In the equation 1, replacing the E with E0 – [ES]

[ ]

Multiplying [S] on both side;

Rearranging ES in denominator,
Placing the value of E0;

As, V0 = [Kcat][ES], So,

Taking –[S] to Km

Rearranging V0,

Scenarios:
As per Michaelis-Menten Equation, when concentration of
[S] is very large, Km can be ignored,
And the equation becomes;

Canceling [S], gives;

When V0=Vmax/2,
Then,

Cancel Vmax, shift 2 to left handside,

Or,

And,

Km is equal to the substrate concentration at which enzyme operates at one half of its maximum
velocity.
TERMINOLOGIES TO KNOW BEFORE

Serine protease: The enzymes that cleave peptide bonds in proteins and have serine that serves
as nucleophilic amino acid at enzyme‟s active site. These enzymes constitute 1/3rd of all
proteolytic enzymes.

Examples: trypsin, elastase, chymotrypsin

These enzymes are secreted by pancreas as an inactive precursor and share similarity in;

 3D structure
 Catalytic triad
 Oxyanion hole (a pocket in the active site of the enzyme that stabilizes the transition
state negative charge on a deprotonated oxygen or alkoxide)
 Covalent acyl-enzyme intermediate

However, differ in their substrate specificity i.e.

Enzyme Substrate Active site composition

Chymotrypsin Aromatic or bulky non-polar Non-polar amino acid
side chains chains
Trypsin Basic residues of amino acids Aspartate (negatively
such as lysine or arginine charged oxygen)
Elastase Smaller and uncharged side No pocket
chains

Chymotrypsin
Bovine pancreatic chymotrypsin is one of the serine proteases that catalyze the hydrolytic
cleavage of peptide bonds.
Properties:

Molecular weight 25.6 kDa

Optimal pH 7.8-8.0
Optimal temperature 40-50°C
Occurrence As a component of pancreatic juice
Activation site Small intestine due to peptidase

Substrate:

Chymotrypsin is selective for peptide bonds with aromatic or large hydrophobic side chains,
such as Tyrosine, Tryptophan, Phenylalanine and Methionine, which are on the carboxyl side of
this bond. It can also catalyze the hydrolysis of ester bond.

Active-site composition:

The main catalytic driving force for the enzyme is the set of three amino acid known as catalytic
triad. This catalytic pocket is found in the whole serine protease family.

Mechanism of action

The catalytic triad Ser/His/Asp acts in a concerted manner and cleaves the peptide bond in two
steps.

1. The acyl group is firstly transferred to Ser hydroxyl oxygen

2. Electron delocalization leading to release of amino group from peptide
3. Addition of water‟s hydrogen to histidine and hydroxyl group to serine residue
4. Electron delocalization leading to release of carboxylic terminal
Biological importance:

 Aid in digestion
 Treat inflammation and reduce swelling (i.e., soft tissue injuries, acute traumatic injuries,
sprains, infections, edema of the eyelids and genitalia, muscle cramps, and sports
injuries)
 Liquefy mucus secretions
 Kill enterozoic worms and other parasites in the digestive tract
 Alleviate effects of chemotherapy
 Act as wound cleaner

Pharmaceutical importance:

When taken orally, inhaled, injected or applied to skin, it is used to treat:

 Ulcers
 Shingles and acne‟
 Surgical or traumatic injuries
 Necrotic tissue
 Help loosen phlegm in asthma, bronchitis, lung diseases, and sinus infections
 Wounds
 Fracture and burn treatments
 Arthritis and such other autoimmune diseases as lupus, scleroderma, and multiple
sclerosis
 Pelvic inflammatory diseases

Side-effects:

 Changes in the color, consistency, and odor of the stool

 Gastrointestinal disturbances such as a feeling of fullness, Diarrhea, Constipation, Nausea
 At high doses, minor allergic reactions like reddening of the skin
 Corneal edema

Ribonuclease
These are a type of nuclease that catalyzes the degradation of RNA into smaller components.

Properties:

It consists of 124 amino acid residues (19 naturally occurring). It operates in an optimum pH
range of 7.0-7.5 and optimal temperature is 40-50°C.

Substrate:
Phosphodiester bond of RNA strand and nucleoside phosphodiester

Active site:

Important residues in the active site are Histidine 12, 119 and Lys 41

Mechanism of action:

RNAs catalyzes the cleavage of the phosphodiester bonds through acid-base catalysis.

The breaking of the bond takes place in two steps;

 1- Formation of the pentavalent phosphate transition state
2- Degradation of the 2'3' cyclic phosphate intermediate

His12 donates a proton to the leaving group of this reaction, the 3' oxygen of the cyclic
intermediate.

Biological importance:

1- RNA degradation
2- Cleaning of cellular RNA that is no longer required
3- Maturation of all RNA molecules
4- Angiogenesis

Pharmaceutical importance:

Treatment of;
1- Allergies
2- Viral infections
3- Tumors
4- Cancers
Induce apoptosis
Eosinophil disorder diagnosis