Enzyme

Enzymes are macromolecular biological catalysts. Enzymes accelerate, or catalyze, chemical reactions. The
molecules at the beginning of the process upon which enzymes may act are called substrates and the enzyme
converts these into different molecules, called products. Almost all metabolic processes in the cell need
enzymes in order to occur at rates fast enough to sustain life. The set of enzymes made in a cell determines
which metabolic pathways occur in that cell. The study of enzymes is called enzymology.
Like all catalysts, enzymes increase the rate of a reaction by lowering its activation energy. Some enzymes can
make their conversion of substrate to product occur many millions of times faster. An extreme example is
orotidine 5'-phosphate decarboxylase, which allows a reaction that would otherwise take millions of years to
occur in milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions,
nor do they alter the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more
specific. Enzyme activity can be affected by other molecules: inhibitors are molecules that decrease enzyme
activity, and activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. An
enzyme's activity decreases markedly outside its optimal temperature and pH
A large protein enzyme molecule is composed of one or more amino acid chains called polypeptide chains. The
amino acid sequence determines the characteristic folding patterns of the proteins structure, which is essential
to enzyme specificity. If the enzyme is subjected to changes, such as fluctuations in temperature or pH, the
protein structure may lose its integrity (denature) and its enzymatic ability. Denaturation is sometimes, but not
always, reversible.
Bound to some enzymes is an additional chemical component called a cofactor, which is a direct participant in
the catalytic event and thus is required for enzymatic activity. A cofactor may be either a coenzymean organic
molecule, such as a vitaminor an inorganic metal ion; some enzymes require both. A cofactor may be either
tightly or loosely bound to the enzyme. If tightly connected, the cofactor is referred to as a prosthetic group.
Like all catalysts, enzymes take part in the reaction - that is how they provide an alternative reaction pathway.
But they do not undergo permanent changes and so remain unchanged at the end of the reaction. They can only
alter the rate of reaction, not the position of the equilibrium.

Most chemical catalysts catalyse a wide range of reactions. They are not usually very selective. In contrast
enzymes are usually highly selective, catalysing specific reactions only. This specificity is due to the shapes of
the enzyme molecules.

Enzymes work in a very unique manner. The way that an enzyme catalyzes a chemical reaction is to start by
building a substrate or several substrates to an active site on the enzyme. This active site is the region on an
enzyme that combines with the substrate. When a substrate and an enzyme bind, the actions cause the
distribution of electrons in chemical bonds of the substrate to change. This eventually leads to a chemical
reaction, which leads to the product.

When the product is made, the enzyme releases it and it begins to regenerate for another cycle of chemical
reactions.
The lock and key hypothesis is focused on the active site. The active site of an enzyme has a very unique
geometric shape and it is only complementary to a specific substrate molecule. Imagine a puzzle piece. There
are only a few pieces that fit with that one piece. Because the active sites are so geometrically unique, an
enzyme can only work with a few or just one substrate.
There are two views regarding the mode of action of enzymes: Lock and Key hypothesis (theory) & Induced fit
hypothesis (theory).
*Lock and Key Theory:* Emil Fisher proposed this hypothesis in [Link] to this hypothesis the active
site of the enzyme is like a lock into which substrate fits like a key.i.e., the shape of the active site and the
substrate molecules are complementary . So the enzyme molecule holds the substrate, molecule close together,
forming the unusable intermediate compound, the enzyme substrate complex. It dissociates to form enzyme and
products.

*Induced Fit Theory:* Daniel [Link] formulated this hypothesis in 1959. According to this hypothesis
the active site does not have a rigid lock and key conformation. The binding of the substrate molecule to the
enzyme molecule induces to modify the shape of the active site so that it becomes complementary to the
substrate molecule. This is called the induced fit . Induced fit is possible because of the flexibility of the protein
molecules.
In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible
structures, the active site is continuously reshaped by interactions with the substrate as the substrate interacts
with the enzyme.[37] As a result, the substrate does not simply bind to a rigid active site; the amino acid sidechains that make up the active site are molded into the precise positions that enable the enzyme to perform its
catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it
enters the active site.[38] The active site continues to change until the substrate is completely bound, at which
point the final shape and charge distribution is determined.[39] Induced fit may enhance the fidelity of molecular
recognition in the presence of competition and noise via the conformational proofreading mechanism

*Lock and Key Theory vs Induced fit Theory*

*Lock and Key Theory:*
1. Active site is a single entity.
2. There is no separate catalytic group.
3. Active site is static.
4. Development of transition state is not considered.
[Link] does not visualize the weakening of substrate bonds.
6. It does not explain the mechanism of non activity in case of
competitive inhibitor.
*Induced fit Theory:*
1. Active site is made of two components.
2. A separate catalytic group is visualized.
3. Active site is not static.
4. It considers the development of transition state before the reacants
undergo change.
5. Catalytic group is believed to weaken the substrate bonds by
nucleophilic and electrophilic attack.
6. It explains the a mechanism for nonaction over competitive inhibitor

Nomenclature
An enzyme will interact with only one type of substance or group of substances, called the substrate, to catalyze
a certain kind of reaction. Because of this specificity, enzymes often have been named by adding the suffix ase to the substrates name (as in urease, which catalyzes the breakdown of urea). Not all enzymes have been
named in this manner, however, and to ease the confusion surrounding enzyme nomenclature, a classification
system has been developed based on the type of reaction the enzyme catalyzes. There are six principal
categories and their reactions: (1) oxidoreductases, which are involved in electron transfer; (2) transferases,
which transfer a chemical group from one substance to another; (3) hydrolases, which cleave the substrate by
uptake of a water molecule (hydrolysis); (4) lyases, which form double bonds by adding or removing a chemical

group; (5) isomerases, which transfer a group within a molecule to form an isomer; and (6) ligases, or
synthetases, which couple the formation of various chemical bonds to the breakdown of a pyrophosphate bond
in adenosine triphosphate or a similar nucleotide.

Acidbase catalysis, acceleration of a chemical reaction by the addition of an acid or a base, the acid
or base itself not being consumed in the reaction. The catalytic reaction may be acid-specific (acid catalysis), as
in the case of decomposition of the sugar sucrose into glucose and fructose in sulfuric acid; or base-specific
(base catalysis), as in the addition of hydrogen cyanide to aldehydes and ketones in the presence of sodium
hydroxide. Many reactions are catalyzed by both acids and bases.
The mechanism of acid- and base-catalyzed reactions is explained in terms of the BrnstedLowry concept of
acids and bases as one in which there is an initial transfer of protons from an acidic catalyst to the reactant or
from the reactant to a basic catalyst. In terms of the Lewis theory of acids and bases, the reaction entails sharing
of an electron pair donated by a base catalyst or accepted by an acid catalyst.
General acid
- partial transfer of a proton from a Brnsted acid lowers the free energy of the transition state rate of reaction increases
with decrease in pH and increase in [Brnsted acid]
Specific acid
- protonation lowers the free energy of the transition state rate of reaction increases with decrease in pH
General base
- partial abstraction of a proton by a Brnsted base lowers the free energy of the transition state rate of reaction increases
with increase in pH and increase in Brnsted base
Specific base
- abstraction of a proton (or nucleophilic attack) by OH
-lowers the free energy of the transition state rate of reaction increases with increase in pH

Mechanism of enzyme action

In most chemical reactions, an energy barrier exists that must be overcome for the reaction to occur. This barrier
prevents complex molecules such as proteins and nucleic acids from spontaneously degrading, and so is
necessary for the preservation of life. When metabolic changes are required in a cell, however, certain of these
complex molecules must be broken down, and this energy barrier must be surmounted. Heat could provide the
additional needed energy (called activation energy), but the rise in temperature would kill the cell. The
alternative is to lower the activation energy level through the use of a catalyst. This is the role that enzymes
play. They react with the substrate to form an intermediate complexa transition statethat requires less
energy for the reaction to proceed. The unstable intermediate compound quickly breaks down to form reaction
products, and the unchanged enzyme is free to react with other substrate molecules.

Only a certain region of the enzyme, called the active site, binds to the substrate. The active site is a groove or
pocket formed by the folding pattern of the protein. This three-dimensional structure, together with the chemical
and electrical properties of the amino acids and cofactors within the active site, permits only a particular
substrate to bind to the site, thus determining the enzymes specificity.

Kinetics

A chemical reaction mechanism with or without enzyme catalysis. The enzyme (E) binds
substrate (S) to produce product (P).

Saturation curve for an enzyme reaction showing the relation between the substrate
concentration and reaction rate.

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data
used in kinetic analyses are commonly obtained from enzyme assays. In 1913 Leonor Michaelis and Maud
Leonora Menten proposed a quantitative theory of enzyme kinetics, which is referred to as MichaelisMenten
kinetics.[58] The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In
the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes
called the Michaelis-Menten complex in their honor. The enzyme then catalyzes the chemical step in the
reaction and releases the product. This work was further developed by G. E. Briggs and J. B. S. Haldane, who
derived kinetic equations that are still widely used today.

Enzyme rates depend on solution conditions and substrate concentration. To find the maximum speed of an
enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen.
This is shown in the saturation curve on the right. Saturation happens because, as substrate concentration
increases, more and more of the free enzyme is converted into the substrate-bound ES complex. At the

maximum reaction rate (Vmax) of the enzyme, all the enzyme active sites are bound to substrate, and the amount
of ES complex is the same as the total amount of enzyme.

Vmax is only one of several important kinetic parameters. The amount of substrate needed to achieve a given rate
of reaction is also important. This is given by the Michaelis-Menten constant (Km), which is the substrate
concentration required for an enzyme to reach one-half its maximum reaction rate; generally, each enzyme has a
characteristic Km for a given substrate. Another useful constant is kcat, also called the turnover number, which is
the number of substrate molecules handled by one active site per second.

The efficiency of an enzyme can be expressed in terms of kcat/Km. This is also called the specificity constant and
incorporates the rate constants for all steps in the reaction up to and including the first irreversible step. Because
the specificity constant reflects both affinity and catalytic ability, it is useful for comparing different enzymes
against each other, or the same enzyme with different substrates. The theoretical maximum for the specificity
constant is called the diffusion limit and is about 108 to 109 (M1 s1). At this point every collision of the enzyme
with its substrate will result in catalysis, and the rate of product formation is not limited by the reaction rate but
by the diffusion rate. Enzymes with this property are called catalytically perfect or kinetically perfect. Example
of such enzymes are triose-phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase,
-lactamase, and superoxide dismutase. The turnover of such enzymes can reach several million reactions per
second.

MichaelisMenten kinetics relies on the law of mass action, which is derived from the assumptions of free
diffusion and thermodynamically driven random collision. Many biochemical or cellular processes deviate
significantly from these conditions, because of macromolecular crowding and constrained molecular movement.
More recent, complex extensions of the model attempt to correct for these effects.

Enzyme inhibitor

An enzyme inhibitor is a molecule that binds to an enzyme and decreases its activity. Since blocking an
enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They
are also used in pesticides. Not all molecules that bind to enzymes are inhibitors; enzyme activators bind to
enzymes and increase their enzymatic activity, while enzyme substrates bind and are converted to products in
the normal catalytic cycle of the enzyme.

The binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or hinder the enzyme
from catalyzing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually
react with the enzyme and change it chemically (e.g. via covalent bond formation). These inhibitors modify key

amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and
different types of inhibition are produced depending on whether these inhibitors bind to the enzyme, the
enzyme-substrate complex, or both.

Irreversible Inhibition: Poisons

An irreversible inhibitor inactivates an enzyme by bonding covalently to a particular group at the active site.
The inhibitor-enzyme bond is so strong that the inhibition cannot be reversed by the addition of excess
substrate. The nerve gases, especially DIFP, irreversibly inhibit biological systems by forming an enzymeinhibitor complex with a specific OH group of serine situated at the active sites of certain enzymes. The
peptidases trypsin and chymotrypsin contain serine groups at the active site and are inhibited by DIFP.
An irreversible inhibitor permanently inactivates the enzyme, usually by forming a covalent bond to the protein
Penicillin[65] and aspirin are common drugs that act in this manner.
Irreversible inhibitors usually covalently modify an enzyme, and inhibition can therefore not be
reversed. Irreversible inhibitors often contain reactive functional groups such as nitrogen
mustards, aldehydes, haloalkanes, alkenes, Michael acceptors, phenyl sulfonates, or
fluorophosphonates. These electrophilic groups react with amino acid side chains to form
covalent adducts. The residues modified are those with side chains containing nucleophiles such
as hydroxyl or sulfhydryl groups; these include the amino acids serine (as in DFP, right), cysteine,
threonine, or tyrosine.

Reversible Inhibition
A reversible inhibitor inactivates an enzyme through noncovalent, more easily reversed, interactions. Unlike an
irreversible inhibitor, a reversible inhibitor can dissociate from the enzyme. Reversible inhibitors include
competitive inhibitors and noncompetitive inhibitors. (There are additional types of reversible inhibitors.)
Reversible inhibitors attach to enzymes with non-covalent interactions such as hydrogen bonds, hydrophobic
interactions and ionic bonds. Multiple weak bonds between the inhibitor and the active site combine to produce
strong and specific binding. In contrast to substrates and irreversible inhibitors, reversible inhibitors generally
do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis.
There are four kinds of reversible enzyme inhibitors. They are classified according to the effect of varying the
concentration of the enzyme's substrate on the inhibitor

Types of inhibition.
In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time, as
shown in the figure on the right. This usually results from the inhibitor having an affinity for the active
site of an enzyme where the substrate also binds; the substrate and inhibitor compete for access to the
enzyme's active site. This type of inhibition can be overcome by sufficiently high concentrations of
substrate (Vmax remains constant), i.e., by out-competing the inhibitor. However, the apparent Km will
increase as it takes a higher concentration of the substrate to reach the Km point, or half the Vmax.
Competitive inhibitors are often similar in structure to the real substrate (see examples below).
In uncompetitive inhibition, the inhibitor binds only to the substrate-enzyme complex. This type of
inhibition causes Vmax to decrease (maximum velocity decreases as a result of removing activated
complex) and Km to decrease (due to better binding efficiency as a result of Le Chatelier's principle and
the effective elimination of the ES complex thus decreasing the Km which indicates a higher binding
affinity).
In non-competitive inhibition, the binding of the inhibitor to the enzyme reduces its activity but does
not affect the binding of substrate. As a result, the extent of inhibition depends only on the concentration
of the inhibitor. Vmax will decrease due to the inability for the reaction to proceed as efficiently, but Km
will remain the same as the actual binding of the substrate, by definition, will still function properly.
In mixed inhibition, the inhibitor can bind to the enzyme at the same time as the enzyme's substrate.
However, the binding of the inhibitor affects the binding of the substrate, and vice versa. This type of
inhibition can be reduced, but not overcome by increasing concentrations of substrate. Although it is

possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from
an allosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this
allosteric site changes the conformation (i.e., tertiary structure or three-dimensional shape) of the
enzyme so that the affinity of the substrate for the active site is reduced.
Examples of reversible inhibitors

As enzymes have evolved to bind their substrates tightly, and most reversible inhibitors bind in the active site of
enzymes, it is unsurprising that some of these inhibitors are strikingly similar in structure to the substrates of
their targets. An example of these substrate mimics are the protease inhibitors, a very successful class of
antiretroviral drugs used to treat HIV.[13] The structure of ritonavir, a protease inhibitor based on a peptide and
containing three peptide bonds, is shown on the right. As this drug resembles the protein that is the substrate of
the HIV protease, it competes with this substrate in the enzyme's active site.

Enzyme inhibitors are often designed to mimic the transition state or intermediate of an enzyme-catalyzed
reaction. This ensures that the inhibitor exploits the transition state stabilising effect of the enzyme, resulting in
a better binding affinity (lower Ki) than substrate-based designs. An example of such a transition state inhibitor
is the antiviral drug oseltamivir; this drug mimics the planar nature of the ring oxonium ion in the reaction of
the viral enzyme neuraminidase.

However, not all inhibitors are based on the structures of substrates. For example, the structure of another HIV
protease inhibitor tipranavir is shown on the left. This molecule is not based on a peptide and has no obvious
structural similarity to a protein substrate. These non-peptide inhibitors can be more stable than inhibitors
containing peptide bonds, because they will not be substrates for peptidases and are less likely to be degraded.
In drug design it is important to consider the concentrations of substrates to which the target enzymes are
exposed. For example, some protein kinase inhibitors have chemical structures that are similar to adenosine
triphosphate, one of the substrates of these enzymes. However, drugs that are simple competitive inhibitors will
have to compete with the high concentrations of ATP in the cell. Protein kinases can also be inhibited by
competition at the binding sites where the kinases interact with their substrate proteins, and most proteins are
present inside cells at concentrations much lower than the concentration of ATP. As a consequence, if two
protein kinase inhibitors both bind in the active site with similar affinity, but only one has to compete with ATP,
then the competitive inhibitor at the protein-binding site will inhibit the enzyme more effectively.

Feedback inhibition

is the phenomenon where the output of a process is used as an input to

control the behavior of the process itself, oftentimes limiting the production of more product. Although negative
feedback is used in the context of inhibition, negative feedback may also be used for promoting a certain
process. An everyday example of negative feedback is the cruise control in automobiles. The faster a car goes
above the cruise control speed, the stronger the brakes are applied to slow the car down. If the car is going to
slowly, more gas is fed to the engine to speed the car up. In a biological context, the more product produced by
the enzyme, the more inhibited the enzyme is towards creating additional product.

Many enzyme catalyzed reactions are carried out through a biochemical pathway. In these pathways, the
product of one reaction becomes the substrate for the next reaction. At the end of the pathway, a desired product
is synthesized. In order to tightly regulate the concentration of that product, the biochemical pathway needs to
be shut down. This is done through feedback inhibition. The product of the final reaction in that pathway reacts
with an enzyme somewhere along the pathway at the enzyme's allosteric site, changing the conformation of the
enzyme. That enzyme can no longer binds to its substrate as effectively due to the conformational change,
closing down that pathway and stopping the final product from synthesizing. The higher the concentration of the
final product, the more likely that product will bind to the allosteric site of the enzyme, shutting down that
pathway.

There are many intermediates and pathways in feedback inhibition. Often the final product Z will inhibit the
initial reactant A.

Mechanism of Negative Feedback

Each metabolic reaction or procesys is regulated by several enzymes. These enzymes control the rate of these
reactions and thus are fundamental in maintaining homeostasis. Below is a universal map of how this type of
inhibition works. We will start with a substrate that is attacked by enzyme 1, forming product A which then acts
as the substrate for enzyme 2 forming product B. Product B then becomes the substrate for the attack of enzyme
3 forming our final product.
substrate ---enzyme 1--> product A ----enzyme 2---> product B ----enzyme 3----> Final Product
Keep in mind that the final product is usually something the body uses up and is necessary for homeostasis. In
this reaction, the purpose of the intermediates, product A and product B, is to move the reaction along to reach
the final product therefore the inhibition mechanism does not start at these intermediates but at the final product.
As the amount of final product becomes elevated, the system imposes a halting effect on enzyme 1, slowing
down the production of intermediates A and B, reducing the formation of the final product. When levels of the
final product fall below a threshold, the effect of negative feedback diminishes and enzyme 1 is reactivated and
the reaction process will be started again.
So what forces are responsible for creating these feedback responses? There are several regulators that affect a
given process. Hormones and chemical signals produced and distributed by the hypothalamus and pituitary
glands, for example, are the regulators that act in feedback loops. To illustrate the concept of this section, let's
investigate the regulation of blood sugar levels. The hormones insulin and glucagon are two regulators that are
intimately related in regulating sugar levels. Insulin is responsible for triggering different cells in the body to
absorb glucose from the blood and to store the excess as glycogen for later utility. Conversely, glucagon's
function is to convert glycogen supply into glucose. When blood glucose level is too low, the alpha cells of the
islets of Langerhans in the pancreas release glucagon. Glucagon subsequently activates the conversion of
glycogen to glucose until the sugar level in the blood is back to its normal state. When blood glucose is too high

the beta cells of the islets of Langerhans release insulin which causes cells in the body to take up sugar quickly,
lowering the blood sugar level to its normal level.

Positive Feedback
In contrast to negative feedback, positive feedback occurs when an output is used as a signal to increase further
response of the output. In other words, if process A results in consequence B, B reinforces process A, resulting
in a cascade where more of B occurs, which causes more of A to occur, and so on. An example of a positive
feedback loop is evolution, where an organism evolves and becomes better at hunting prey, for example, prey
evolve better defense mechanism like faster running, which causes predators to adapt by evolving better chasing
skills, and so forth. Note that a positive or negative feedback mechanism is not necessarily beneficial or
harmful; they only refer to the mechanism by which inhibition or propagation occurs.