Department of Botany

Mid – Term Assignment

Course code: CHM-5104 Course name: Biochemistry

Due Date: 04-20-2020 Submission Date: 04-20-2020

Course Teacher: Sir Rana Mahmood Student Name: Wajeeh un Nisa

Course ID: BBOF18E027 Session: 2018-2022

Program: BS Botany Semester: 4th

Regular/ Self Support: Self Support Main Campus/ PPP: Main Campus

ASSIGNMENT COVER SHEAT

Title of Assignment: Mechanism of Enzyme Action

Word Count: 3069

Marks awarded by Teacher

Enzyme:

• They are biocatalysts which accelerate the chemical reactions.

• They are protein in nature.
• They are protein in nature.
• They have low energy of activation.
• All the food digests in our body is due to the enzymes action.

Enzyme mechanism:

• The chemical transformation, and all steps occur within them, generated
by enzymatic action on substrates.
• So, all the steps and actions generated on substrate by enzymes all called
enzymes action.
Substrate:

• A solid substance or medium to which another substance is applied and

second substance bind like enzyme which bind on it for biochemical
reactions.
• After chemical reaction substrate change into product but enzyme does not
change. Substrate change but enzyme does not change and specific for each
substrate molecule.
• After the enzyme action on the substrate break substrate into many products.

Active site:

• The active site is place where chemical reactions take place.

• There substrate molecule attach and enzyme also act on it and on active site
substrate change into product.
• The active sit consists of amino acid and residues that form temporary bonds
with the substrate and residues that catalyse a reaction of that substrate.
Activation energy:

• The minimum amount of energy that is required to activate atoms or

molecules to a condition in which they undergo chemical transformation.
• This energy is required to change reactant into product. But the amount of
energy should be minimum.

Inhibitors:

• A substance that bind to enzyme and decrease its activity .

• By binding to enzyme active sites, inhibitors reduce the compatibility of
substrate and enzyme.
• They slow the chemical reactions and sometimes stop the chemical reaction.
• They are also called the substance which reduces or suppresses the activity of
another substance.

Types:

There are two types of inhibitors

1: Reversible inhibitors

2: Irreversible inhibitors
Reversible Inhibitors:

• They stop the rate of reactions by occupying the active sites.

• They destroy the globular structure of enzyme by permanently occupying
the active sites.
• They form the covalent bond with the active site which is permanent and
destroy the structure of enzyme.
• They physically block the active site and it will unable to attach any
substrate and do any reaction.
• Enzyme permanently denatured.

Irreversible Inhibitors:

• They are like substrate and occupy the active site temporarily.
• They form weak linkage with substrate.
• They break the bond with the active site when the amount of the substrate
is increased.
• The greater amounts of the substrate force the inhibitor to break bond with
active site.

Types of Reversible Inhibitor:

There are two types of inhibitors:

(a) Competitive Inhibitor

(b) Non Competitive Inhibitors

(a) Competitive Inhibitor:

• They have structural similarity with substrate.
• They bind at the active site because active site do not recognize the
inhibitor molecule due to similarity with the substrate molecule.
• The inhibitor is not able to activate the catalytic site.
• Thus, active site is not able to change inhibitor in to product.
 (b) Non Competitive Inhibitor:
• They form enzyme inhibitor complex at a point other than active site.
• They do not bind exactly at active site.
• They totally change the structure of enzyme.
• They change structure of enzyme in such a way that if actual substrate
binds at active site catalysis will not occur.
• It retarded the process of catalysis in the enzyme molecule.
• The enzyme structure will denatured and made it unable for catatalysis.

Activator:

• The molecules which increase the activity of enzyme are activator.

• The detachable co- factor is activator.
• It is an inorganic ion.
Co factor:

• Some cofactors contain non protein part called co-factor.

• It is essential for the proper functioning of the enzymes.
• It is bridge between the enzyme and the substrate.
• It takes part in chemical reactions.
• It brings about catalysis.
• It plays a role of chemical energy and brings about the reactions which are
not possible to occur.
• The co-factors bring about catalysis and increase the rate of reaction.
• Some co factors are used as metal ion by enzymes.
• Enzyme increases the rate of reaction but catalyst also increase the rate of
enzyme reactions.
• Enzymes rate of reaction increase in the presence of cofactor

Structure of enzyme:

• They are globular proteins, acting alone or in large complexes.

• The sequence of the amino acids specifies the structure which in turn
determines the catalytic activity of enzymes.
• Enzyme structure is denatured by extreme temperature and PH.
• They are much large in size than their substrate.
Factors effecting enzyme activity:

There are following factors that effect the activity of enzyme.

Temperature:

• Enzymes activity increases as temperature increases to a certain limit.

• Enzymes work at optimum temperature.
• The optimum temperature of our body is 37. So, enzymes work more
efficiently at optimum temperature.
• Heat provides activation energy and therefore, chemical reactions are
accelerated at high temperatures.
• Heat also supplies kinetic energy to the reacting molecules, causing them
to move rapidly.
• Thus, the reactants movie more quickly and chances of their collision
with other increases
• However, if temperature increases more than limit the vibration of the
molecules increases and their structure denature.
• But if temperature increases more than 37 the enzymes do not work
efficiently and get denatured.
• So, optimum temperature is necessary for enzymes to work efficiently.

PH:
 • Every enzyme function more efficiently over a narrow range of PH known
as optimum PH.
• Enzymes work more efficiently at optimum PH.
• A slight change in PH can change the ionization of the amino acids at the
active site.
• Moreover, it effects the concentration of the substrates.
• When such condition occurs then enzymes stop working and enzymes get
denatured.

Enzyme concentration:

• The rate of reaction is effected by the enzyme concentration present at

specific time at unlimited substrate concentration.
• The reaction rate becomes double if enzyme concentration increases.
• The enzymes speed up the chemical reaction and their more concentration
increases rate of reaction because the active site of the enzymes increases
and more active site convert more substrate into a product.

Substrate Concentration:
 • At low concentration of Substrate rate of reaction is directly proportional
to the amount of the substrate available.
• If the amount of substrate increases but the enzyme concentration kept
constant then at a point reach when further increase in the substrate
concentration does not increase rate of reaction.
• This is due to all the active sites of the enzymes are occupied by the
substrate concentration and reaction rate does not increase.
• Thus more the amount of substrate concentration does not increase rate of
reaction if amount of enzyme concentration put constant.

Mechanism of enzyme action:

• Any enzyme react with specific substrate and change it into product and
enzyme remain unchanged.
• The enzyme is used again and again more the substrate is added and more
reactants will change into product.

• In the catalysis process substrate in the presence of enzyme reacts with

each other on a charges active site and change substrate into substrate
complex and then product formed and enzyme remain unchanged.
 • In some cases enzymes react with substrate in a series where enzymes
remain same but amount of substrate is added and product form in series.
• The reactions that occur in series are metabolic pathways.
• The enzymes bring about respiration and photosynthesis process.
• The product from one step in pathway is transferred to enzyme catalyzing
in the next step

• In all process enzymes are reused.

• The all reaction of the catalysis of occurs in human body by floating the
cytoplasmic soup in the cell.
• But some enzymes do not float in the cytoplasmic soup and they attached
with the cell membrane from inside in a specific and orderly
arrangements.
• In mitochondria and chloroplast they do not in the cytoplasmic soup but
attach with the cell membrane.

Substrate binding:

• Before the enzymes start their biochemical reaction they must bind their
substrate.
• Enzymes are specific in their action.
• For each substrate molecule one enzyme present which start the
biochemical reaction. For example, for urea urease enzyme is present.
• The specificity is achieved by binding pockets shaper, charge and
hydrophobic characteristics to the substrate.
• Enzyme therefore can be distinguished between very similar substrate
molecules to be chemoselective, regioselective and stereospecific.
• Some of the enzymes showing the highest specificity and accuracy are
involved in copying and expression of the genomes.
• Some of these enzymes are proof reading mechanisms. Here, an enzyme
such as DNA polymerase catalyses a reaction in a first step and then checks
that product is correct ion a second step.
• Conversely, some enzymes display enzyme promiscuity, having broad
specificity and acting on a range of different physiologically relevant
substrates.
• Many enzymes possess small side activities which arose neutrally.
• Enzyme molecule usually has two active sites where specific substrate
binding occur.
• The substrate bind on the active sites because active sites contain binding
sites where substrate attach and binding site orient it for catalysis.
• Initially active site and substrate are not covalently bonded.
• The four type of bonding present between the active site and the substrate
complex. These bonds are following:

Hydrogen bonds

Vander waals interactions

Hydrophobic interactions

Electrostatic force interactions

• The charge present on the active site is neutral means no positive or

negative charge is present.
• If charge present on them repulsive force will push them because if same
charge is present on the active site and substrate no binding will occur and
it push them apart.
• On the active site usually non polar amino acids are present but sometimes
polar amino acids also present which carry either negative or positive
charges.
• Enzymes are protein in nature and protein contain 3 structures primary,
secondary, and tertiary but for the enzyme functions enzyme need tertiary
structure.
• To maintain three dimensional structure, proteins depends on various types
of interactions present among their amino acids.
• The external and internal environment changes like PH more or less than 7,
temperature of body more than 37 or concentration of ions increases then
protein structure become denature and interaction among the amino acids
also ended and enzymes lose their catalytic activity.
• If the binding between the amino acid and active site is strong the catalysis
occurs more efficiently.
• The binding site helps the enzyme for the recognition and binding of the
substrate.
• Thus substrate complex is formed.
• The reaction activates the catalytic site to do more reactions.
• Activated catalytic site change the enzyme substrate complex into a
product and enzyme remain unchanged during this process.
• Enzyme detaches from the product formed and do more reactions and used
again and again.
Models which show how enzymes fit their specific substrate:

• Lock and Key Model

• Induced Fit Model
• The Conformational Selection Model
• Substrate Strain Theory

Lock and Key Model:

• To observe the specificity of enzymes, Email Fisher proposed Lock and Key
model.
• He proposed that the active site and substrate are two stable structures that
fit perfectly without any changes, just like key fit into the lock.
• According to Lock and Key model both enzymes and substrate possess
specific complementary geometric shapes that fit exactly into one an other.
• According to Lock and Key Model active site is a rigid structure which does
not change its shape according to enzyme.
• But enzyme is flexible and changes its shape according to active site.
• Thus enzyme is not stable structure.
• If one substrate strongly bond with the active site the interaction among
them will strong and show high catalytic activity.
• With the time limitations of this model appear.
• For example, the competitive enzyme inhibitor methylglucoside can bind
tightly to the active site of 4- alpha glucanotransferase and perfectly fits into
it.
• However, 4- alpha-glucanotransferase is not active on methylglucoside and
no glycosyl transfers occur.
• The Lock and Key Model just explain competitive inhibitors and not explain
non competitive inhibitors because non competitive inhibitors not bind to
active site which start catalytic activity.
Induced Fit Model:

• Daniel Koshland suggested a modification in the Lock and Key Model and
proposed Induce Fit Model.
• He said that enzyme is a flexible structure but active site is not a rigid
structure it also flexible structure and change its shape according to
substrate.
• As a result enzyme, the substrate does not simply bind to a rigid active sit;
the amino acid side-chains that made the active site change into precise
positions that enable that enable enzyme to perform its catalytic function.
• Some substrates when enter the active site also change its shape according
to active site such as glycosidases, which change its shape according to
active site.
• The active site change its shape until the substrate molecule not fit into the
active site.
• Daniel Koshland’s theory of enzyme substrate binding is that the active site
and binding portion of the substrate are not exactly complementary.
• This model is similar to a person wearing gloves: the gloves changes shape
to fit the hand.
• The enzyme initially has a conformation that attracts its substrate.
• Enzyme is a flexible structure and also active is and only correct catalyst
can induce interaction leading to catalysis.
• Conformational changes may then occur .as the substrate is bound.
 • When the product is formed it move away from the enzyme and active site
will gain its original position.
• Some hypothesis that were proved by observations said that protein move
during catalysis which support the catalysis process.

Conformational Selection Model:

• This model says that change in enzyme shape occurs.

• Protein may not wholly follow this model.
• Amino acids at the binding site generally follow Induce Fit Model,
whereas those proteins who left follow the Conformational Selection
Model.
• Factors such as temperature effect during binding, when temperature is
high it increase Conformational Selection Model and decreases Induce
Fit Model.
• This model suggests that enzymes exist in a variety of conformations,
only which some are able to binding with substrate.
• When a substrate bind with enzyme, the equilibrium in the
conformational ensemble shifts towards those able to bind ligands.

Substrate Strain Theory:

• According to this theory after the bonding of the substrate with the active
site the enzyme which speeds up the chemical reaction produces a strain
to the substrate.
 • The substrate strain formation becomes the reason of the product
formation.
• The substrate formation speed up the chemical reaction
• The binding of the substrate to the active site produce damage to the
substrate because substrate has to change its shape to fit in the active site
for the reaction and thus product formation occurs.

Mechanism of Enzyme Catalyzed reactions:

• The enzymes speed up the chemical reaction inside the human body but
when enzymes are catalyzed by the metal ion or other catalyst it speed up
its reaction 6 to 12 time as compared to non catalyzed chemical reaction.
• This increase in the rate of the chemical reaction with such a speed is due
to the four processes.
(1)Acid Base catalysis

(2)Substrate strain catalysis

(3)Covalent Catalysis

(4)Entropy Effect

(1)Acid Base catalysis:

• The reaction rate increases if the acid or base is added to it but the acid
base just speed up the chemical reaction but not use itself and after the
product formed acid and base detaches and again used in the other
chemical reaction.
• The reaction may be acid specific or base specific but both together are
not used in the chemical reaction

For Example;

• The decomposition of sucrose into the glucose and fructose is acid

catalysis because it occurs in the presence of the sulfuric acid.
 • And the addition of hydrogen cyanide to aldehydes and ketones is base
catalysis because it occurs in the presence of the sodium hydroxide.
✓ Some reactions have both acid and base catalysis occurs.
✓ Their catalysis mechanism is explained by the Bronsted-
Lowary concept.
✓ According to this concept initial transfer of proton occurs
from reactant to acid catalyst or base catalyst.

(2) Substrate Strain Catalysis:

• The increase in the rate of the chemical reaction takes place by the active
site of the protein.
• The substrate form bond with the active site and enzyme produces a
strain as a result substrate strain bond is formed and substrate changes
into the product.
• The change in the shape of the substrate is produce due to active site and
thus product formation takes place.
• Thus, this substrate strain increases the rate of the chemical reaction.
(3) Covalent Catalysis:

• In such type of catalysis there is a covalent bond formed between the

substrate active site of the enzyme or with a cofactor which is a non
protein.
• The substrate becomes temporarily covalently attached to enzyme during
catalytic reaction.
• The nucleophile is highly reactive so enzymatic reactions contains
nucleophilic residue which react with the substrate to change it into a
product.
• Electrophonic is withdrawal of electron from the reaction centre.
• The electrophilic reaction takes place between the substrate and catalyst.

Entropy Effect:

• In the rate of chemical reaction when temperature of the any substance

increases the molecular motion also increases and entropy occurs.
• The decrease in the temperature during a chemical reaction decrease the
molecular motion of the substances and entropy also decreases.
• So, we can say entropy increases during exothermic reactions and decreases
during endothermic reactions.
• Thus reaction rate increases when temperature increases and entropy also
increases.
References:

1 Callahan BP, Miller BG (December 2007). OMP decarboxylase _An enigma

persists. Bioorganic Chemistry. 35(6): 465_9.

Dio:10.1016/j.bioorg.2007.07.004.

PMID 17889251.

2 Williams HS(1904). A History of Science: in Five Volumes. Volume iv:Modern

Development of the Chemical and biological Sciences. Harper and Brothers.

3 Eduard Buchner _Nobel Lecture: Cell _Free Fermentation. Nobel

prize.org.1907. Retrieved 23 February 2015.

4 Eduard Buchner. Nobel Laureate Biography. Nobelprize.org. Retrieved 23

February 2015.

5 https://en.m.wikipedia.org>wiki

6 From FSC Book of Biology Part(1)

(7) https://britannica.com