Enzyme catalysis. kinetics of enzyme

1. ENZYME KINETICS
CONTENTS
• Introduction
• Enzymes
• Nature of enzymes
• Enzyme kinetics
• Working of enzyme
• Mechanism of enzyme
• Lock and key model
• Induced fit model
• Factors affecting rate of enzyme action
• Temperature
• pH
• Substrate concentration

2. INTRODUCTION
The existence of enzymes has been known for well over a century. Some of the earliest studies
were performed in 1835 by the Swedish chemist Jon Jakob Berzelius who termed their chemical
action catalytic. It was not until 1926, however, that the first enzyme was obtained in pure form,
a feat accomplished by James B. Sumner of Cornell University. Sumner was able to isolate and
crystallize the enzyme urease from the jack bean. His work was to earn him the 1947 Nobel
Prize. John H. Northrop and Wendell M. Stanley of the Rockefeller Institute for Medical
Research shared the 1947 Nobel Prize with Sumner. They discovered a complex procedure for
isolating pepsin. This precipitation technique devised by Northrop and Stanley has been used to
crystallize several enzymes.
ENZYMES
Enzymes are the biological catalysts that can accelerate a specific chemical reaction by lowering
the activation energy but remain unaltered in the process.
The living cell is the site of tremendous biochemical activity called metabolism. This is the
process of chemical and physical change which goes on continually in the living organism.
Build-up of new tissue, replacement of old tissue, conversion of food to energy, disposal of
waste materials, reproduction - all the activities that we characterize as "life." This building up
and tearing down takes place in the face of an apparent paradox. The greatest majority of these
biochemical reactions do not take place spontaneously. The phenomenon of catalysis makes
possible biochemical reactions necessary for all life processes. Catalysis is defined as the
acceleration of a chemical reaction by some substance which itself undergoes no permanent
chemical change. The catalysts of biochemical reactions are enzymes and are responsible for
bringing about almost all of the chemical reactions in living organisms. Without enzymes, these
reactions take place at a rate far too slow for the pace of metabolism. The oxidation of a fatty
acid to carbon dioxide and water is not a gentle process in a test tube - extremes of pH, high
temperatures and corrosive chemicals are required. Yet in the body, such a reaction takes place
smoothly and rapidly within a narrow range of pH and temperature. In the laboratory, the
average protein must be boiled for about 24 hours in a 20% HCl solution to achieve a complete
breakdown. In the body, the breakdown takes place in four hours or less under conditions of mild
physiological temperature and pH.
Nature Of Enzyme
All known enzymes are proteins. They are high molecular weight compounds made up
principally of chains of amino acids linked together by peptide bonds. Enzymes can be
denatured and precipitated with salts, solvents and other reagents. They have molecular
weight ranging from 10,000 to 2,000,000.

3. Many enzymes require the presence of other compounds - cofactors - before their
catalytic activity can be exerted. This entire active complex is referred to as the
holoenzyme; i.e., apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic
group or metal-ion- activator) is called the holoenzyme.
Apoenzyme + Cofactor = Holoenzyme
According to Holum, the cofactor may be:
1. A coenzyme - a non-protein organic substance which is dialyzable, thermostable and loosely
attached to the protein part.
2. A prosthetic group - an organic substance which is dialyzable and thermostable which is
firmly attached to the protein or apoenzyme portion.
3. A metal-ion-activator - these include K+ , Fe++ , Fe+++ , Cu++ , Co++ , Zn++ , Mn++ ,
Mg++ , Ca++ , and Mo+++ .
ENZYME KINETICS
It is the study of chemical reactions catalysed by enzymes.
Working of Enzyme
Enzymes help to speed chemical reactions by lowering activation energy, which is the energy
required for a chemical reaction to occur. When enzymes are present, this energy requirement is
lowered, allowing the reaction to occur more quickly. Let's have a look at an example of this:

4. This graphic has two lines showing the reaction rates with enzymes and without enzymes . The
dotted line represents the activation energy needed for this reaction to occur. Notice how there is
less required energy in the enzyme driven reaction. The enzyme allows the reaction to occur
more quickly than the reaction without an enzyme. Enzymes are highly useful, however, their
reaction rates can be altered by their surroundings.
MECHANISM OF ENZYME ACTION
Enzyme Characteristics:
The basic mechanism by which enzymes catalyze chemical reactions begins with the binding
of the substrate (or substrates) to the active site on the enzyme. The active site is the specific
region of the enzyme which combines with the substrate. The binding of the substrate to the
enzyme causes changes in the distribution of electrons in the chemical bonds of the substrate
and ultimately causes the reactions that lead to the formation of products. The products are
released from the enzyme surface to regenerate the enzyme for another reaction cycle.
The active site has a unique geometric shape that is complementary to the geometric shape of a
substrate molecule, similar to the fit of puzzle pieces. This means that enzymes
specificallyreact with only one or a very few similar compounds.
Lock and Key Theory:
The specific action of an enzyme with a single substrate can be explained using a Lock and Key
analogy first postulated in 1894 by Emil Fischer. In this analogy, the lock is the enzyme and the
key is the substrate. Only the correctly sized key (substrate) fits into the key hole (active site) of
the lock (enzyme).

5. Smaller keys, larger keys, or incorrectly positioned teeth on keys (incorrectly shaped or sized
substrate molecules) do not fit into the lock (enzyme). Only the correctly shaped key opens a
particular lock.
Induced Fit Theory:
Not all experimental evidence can be adequately explained by using the so-called rigid enzyme
model assumed by the lock and key theory. For this reason, a modification called the induced-fit
theory has been proposed.This model is proposed by Daniel Koshland in1958.
The induced-fit theory assumes that the substrate plays a role in determining the final shape of
the enzyme and that the enzyme is partially flexible. This explains why certain compounds can
bind to the enzyme but do not react because the enzyme has been distorted too much. Other
molecules may be too small to induce the proper alignment and therefore cannot react. Only the
proper substrate is capable of inducing the proper alignment of the active site.
In the graphic on the left, the substrate is represented by the magenta molecule, the enzyme
protein is represented by the green and cyan colors. The cyan colored protein is used to more
sharply define the active site. The protein chains are flexible and fit around the substrate.
FACTORS INFLUENCING RATE OF ENZYME ACTION

6. There are several factors that can influence the rate of enzyme reactions. The most common
include changes to pH, temperature, and substrate concentration.
Temperature
Collisions between all molecules increase as temperature increases. This is due to the increase in
velocity and kinetic energy that follows temperature increases. With faster velocities, there will
be less time between collisions. This results in more molecules reaching the activation energy,
which increases the rate of the reactions. Since the molecules are also moving faster, collisions
between enzymes and substrates also increase.
Optimum Temperature
Each enzyme has a temperature that it works optimally in, which in humans is around 98.6
degrees Fahrenheit , 37 degrees Celsius – the normal body temperature for humans. However,
some enzymes work really well at lower temperatures like 39 degree Fahrenheit, 4 degrees
Celsius, and some work really well at higher temperatures. For instance, animals from the Arctic
have enzymes adapted to have lower optimum temperatures while animals in desert climates
have enzymes adapted to higher temperatures. While higher temperatures do increase the activity
of enzymes and the rate of reactions, enzymes are still proteins, and as with all proteins,
temperatures above 104 degrees Fahrenheit, 40 degrees Celsius, will start to break them down.
So, the two ends of the activity range for an enzyme are determined by what temperature starts
the activity and what temperature starts to break down the protein.

7. pH
The pH scale runs from 1 - 14 and is a measure of how acidic or basic a substance is. For
example, something with a pH value of 2 represents a strong acid, such as lemon juice.
Conversely, a pH value of 13 represents a strong base, such as bleach. Outside their optimal
ranges, enzymes work less effectively.
The pH of a solution can have several effects of the structure and activity of enzymes.
For example, pH can have an effect of the state of ionization of acidic or basic amino acids.
Acidic amino acids have carboxyl functional groups in their side chains. Basic amino acids
have amine functional groups in their side chains. If the state of ionization of amino acids in a
protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be
altered. This can lead to altered protein recognition or an enzyme might become inactive.
Changes in pH may not only affect the shape of an enzyme but it may also change the shape or
charge properties of the substrate so that either the substrate connot bind to the active site or it
cannot undergo catalysis.
In geneal enzyme have a pH optimum. However the optimum is not the same for each enzyme.
CONCENTRATION
• Changing the Enzyme and Substrate concentrations affect the rate of reaction of an
enzyme-catalysed reaction. Controlling these factors in a cell is one way that an
organism regulates its enzyme activity and so its Metabolism.
• Changing the concentration of a substance only affects the rate of reaction if it is
the limiting factor that is, it the factor that is stopping a reaction from preceding at
a higher rate.

8. • If it is the limiting factor, increasing concentration will increase the rate of reaction up to
a point, after which any increase will not affect the rate of reaction. This is because it
will no longer be the limiting factor and another factor will be limiting the maximum
rate of reaction.
• As a reaction proceeds, the rate of reaction will decrease, since the Substrate will get used
up. The highest rate of reaction, known as the Initial Reaction Rate is the maximum
reaction rate for an enzyme in an experimental situation.
Substrate concentration
• Increasing Substrate Concentration increases the rate of reaction. This is because more
substrate molecules will be colliding with enzyme molecules, so more product will be
formed.
• However, after a certain concentration, any increase will have no effect on the rate of
reaction, since Substrate Concentration will no longer be the limiting factor.
The enzymes will effectively become saturated, and will be working at their maximum
possible rate.
Enzyme concentration
• Increasing Enzyme Concentration will increase the rate of reaction, as more enzymes will
be colliding with substrate molecule.
• However , this too will only have an effect up to a certain concentration, where the
enzyme concentrationis no longer the limiting factor .