Enzymatic Test Reaction

ENZYMES
In 1833, a French chemist Anselme Payen was the first to discover the vital force that drove these
reactions and named it “ENZYME”.

ENZYMES are substances, proteins or in some cases ribonucleic acid (RNA), that speed up a
biochemical reaction by modifying specific substances called substrate. Enzymes are supremely selective
in who they bind and modify, hence their “specificity”. Human Body is a product of different chemical
reactions and processes.
“But what controls these reactions?”

What is enzymatic test reaction?

An enzyme attracts substrates to its active site, catalyzes the chemical reaction by which
products are formed, and then allows the products to dissociate or separate from the enzyme surface.

What happens to an enzyme during enzymatic reaction?

To catalyze a reaction, an enzyme will grab on or bind to one or more reactant molecules.
A substrate enters the active site of enzyme and the substrate form a temporary complex.
example
Enzymes have a specific place in them called “Active Site” where the substrate binds and
real time action takes place. Active site has a specific size, shape and chemical behavior rendered
(yield, breakdown or melt) to it by specific arrangement of amino acids. Thanks to these amino
acids, an enzymes active site is unique only to a particular substrate. In addition to the active site,
many enzymes consist of a non-protein part called “Cofactor”.

Cofactors may be:

Cations: Positively charge metal ions (activators), bound temporarily to the active site to “active” the
enzymes.

Organic molecules: Vitamins or vitamin products (coenzymes) that join enzyme-substrate complex
temporarily.

Prosthetic groups: Permanently enzymes bound.

Many enzymes only perform their catalytic role when associated a coenzyme. And an in activated enzyme
(protein) along with its coenzyme (non-protein) makes up a system called holoenzyme. To add to the
complexity, scientist termed this inactivated enzyme as apoenzyme and therefore the equation becomes:
Holoenzymes= Apoenzyme+ Coenzyme.

How does pH affect enzymatic reaction rate?

The closer the pH is to 7, the higher the reaction rate was due to the distance from the
optimum. However, the reaction decreases because the space of the enzyme active began to deform.
 Enzyme examples and principles:

A researcher is studying the rate of an enzyme-catalyzed reaction by placing increasing amounts

of substrate into a solution containing the enzyme. After a certain concentration, the rate of the
reaction plateaus and does not go any higher. What has happened?

The answer is: the enzyme has become saturated.

Explanation:
If the reaction rate has plateaued, this indicates that the enzyme has reached saturation. At this
point, every active site on every molecule of enzyme is actively catalyzing the reaction as quickly as it
can. The only way to change the reaction rate, at this point, would be to increase the concentration of
the enzyme in the solution. Further increasing substrate concentration will have no effect.

A catalyst is an enzyme that promotes a reaction. In terms of free energy, what does a catalyst change
about the reaction to promote the reaction proceeding?

The answer is: catalysts increase the rate of the reaction by reducing the free energy of the transition
state, which lowers the activation energy.

Explanation:
During a reaction, the reactants must pass through high-energy transition states before they
evolve into the products. The catalyst reduces the free energy of this transition state, thus making it
'easier' for the reactant to undergo the chemical reaction since the activation energy has been lowered.

MODELS OF ENZYME ACTION

Lock and key hypothesis

This is simplest model to represent how enzyme works. The substrate simply fits into the active
site to form a reaction intermediate just like the key fits in its specific lock. The shape isn’t changed here,
rather the structure of substrate absolutely compliments the structure of the enzymes, like puzzle quizzes.

Induced fit hypothesis

In this model, the enzyme, upon binding of substrate, changes shape. The matching between an
enzyme’s active site and the substrate isn’t just like two puzzle pieces fitting together, rather the enzyme
changes shape and binds to its substrate even more tightly. This fine-tuning of the enzyme to “fit” the
substrate is called induced fit.
Factors Affecting Enzymatic Reaction Concentration of Enzyme

As the concentration of the enzyme is increased, the velocity of the reaction proportionately
increases. This property is used for determining the activities of serum enzymes during the
diagnosis of disease.

Increasing enzyme concentration will increase the rate of reaction, as more enzymes will be
available to bind with substrates. However, after a certain concentration, any increase will have
no effect on the rate of reaction.

Concentration of Substrate

In the presence of a given amount of enzyme, the rate of enzymatic reaction increases as the
substrate concentration increases until a limiting rate is reached, after which further increase
in the substrate concentration produces no significant change in the reaction rate. At this
point, so much substrate is present that essentially all of the enzyme active sites have
substrate bound to them.
In other words, the enzyme molecules are saturated with substrate. The excess substrate
molecules cannot react until the substrate already bound to the enzymes has reacted and
been released or been released without reacting.

Increasing substrate concentration increases the rate of reaction. This is because more
substrate molecules, will be colliding with enzymes molecules, so more product will be formed.
But again, this affect is valid up to a certain concentration.

Effect of Temperature
Raising the temperature increases the rate of both un-catalyzed and enzyme-catalyzed
reactions by increasing the kinetic energy and the collision frequency of the reacting
molecule.

The polypeptide chain that begins to unfold, or denature with an accompanying loss of
catalytic activity. The temperature range over which an enzyme maintains a stable,
catalytically competent information depends upon and typically moderately exceeds the
normal temperature of the cells in which it resides.

The Temperature Coefficient (Q10) is the factor by which the rate of a biologic process
increases for a 10°C increase in temperature. For the temperatures over which enzymes are
stable, the rates of most biological processes typically double for a 10°C rise in temperature
(Q10=2).

The suitable temperature for enzymes to function properly is 37°C. Increasing or decreasing
the temperature above 37°C affect chemical bonds in the active site, making them less suited
 to bind substrates. Higher temperature denature enzymes.

Effect of pH

Enzymes are protein substances that contain acidic carboxylic groups (COOH–) and basic
amino groups (NH2). So, the enzymes are affected by changing the pH value.
Each enzyme has a pH value that it works at with maximum efficiency called the optimal pH.
If the pH is lower or higher than the optimal pH, the enzyme activity decreases until it stops
working.

Amino acids present in the active site are acidic or basic. Fluctuation (changes/inconstancy) in
PH can affect these amino acids, making it hard for substrate to bind. Extreme pH values can
denature enzymes.

Effect of Inhibitors on Enzyme Activity

Enzyme inhibitors are substances which alter the catalytic action of the enzyme and
consequently slow down, or in some cases, stop catalysis. There are three common types of
enzyme inhibition - competitive, non-competitive and substrate inhibition.

Some harmful substances, called “Inhibitors”, reduce or even stop the activity of enzymes in
biochemical reactions. They either block or distort the active site, thus inhibiting the reaction.
Based on this, there are two types of inhibitors given below;
Competitive Inhibitor
 The effects of competitive inhibitors can be overcome by raising the concentration
of substrate. Most frequently, in competitive inhibition the inhibitor (I) binds to the
substrate-binding portion of the active site thereby blocking access by the
substrate. A competitive inhibitor acts by decreasing the number of free-enzymes
molecules available to bind substrate that is to form ES and thus eventually to form
a product.

 Occupy the active site and prevent a substrate molecule from binding to the
enzyme.

Non-competitive Inhibitor

 Non-competitive inhibition binding of the inhibitor does not affect binding of

the substrate. Formation of both EI and EIS complexes is therefore possible.

 Attach to parts of the enzyme, other than the active site to distort the shape of
an enzyme.

Substrate Inhibitor

 Substrate inhibition will sometimes occur when excessive amounts of substrate

are present.
Reference:

Retrieve from:

http://www.worthington-biochem.com/introBiochem/enzymeConc.html

https://alevelbiology.co.uk/notes/factors-affecting-enzyme-activity/

Harper's illustrated biochemistry 30th edition