CHEMISTRY-VIII NOTES PREPARED BY Dr.

DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

UNIT-III
Enzymes 4 hours Max. Marks: 10
Introduction, holoenzyme (apo enzyme and co-enzyme). Active site, specificity
(Group, absolute and stereo selectivity with examples). Classification of enzymes
(EC code number not required) with examples. Enzyme substrate interaction-
Fischer and Koshland models. Enzyme kinetics - factors affecting rate of
enzymatic reactions - enzyme concentration, substrate Concentration (mention M.
M. equation), pH and temperature . Allosteric enzymes - definition and example
Enzyme inhibitions-Competitive, noncompetitive and uncompetitive inhibition
with one example for each.

Introduction: Enzymes are molecules that catalyze biochemical reactions. They

usually exist in very low concentrations in cells, where they catalyze metabolic
reactions. They are the functional units of cell metabolism. They act in organized
sequences, catalyzing the hundreds of stepwise reactions of catabolism and
anabolism. Their highly regulated and coordinated action sustains the living state.
Enzymes may be simple proteins, in which only the native conformation of the
protein is required for activity. If the enzyme is conjugated protein, their activity
will depends upon the protein of native conformation and additional small
substance called cofactors. A cofactor may be defined as a low molecular weight,
heat-stable, inorganic or organic substance required for the catalytic function of an
enzyme.
Cofactors may be roughly categorized into i) simple metal ions ii) coenzymes
and iii) prosthetic groups.
Examples of metal ion cofactors are zinc of carboxypeptidase A, copper of lysine
oxidase, Mg2+ of hexokinase

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

Coenzymes: The complex organic substances other than protein that acts together
with the enzyme to catalyze a biochemical reaction are called coenzymes. Most of
the coenzymes are modified forms of vitamins.
Prosthetic group: The non –protein part of the conjugated proteins which are
tightly bound to the enzyme and are required for their catalytic activity is called
prosthetic group
Example: Porphyrin of haemoglobin and peroxidase.
Coenzymes may also be prosthetic group.
Example: Flavin Adenine Dinucleotide of succinic dehydrogenase.
Apoenzyme: The protein part of the enzyme without the cofactor(coenzyme or
prosthetic group) which catalytically inactive is called the apoenzyme.
Holoenzyme: A catalytically active enzyme associated with its cofactor is called
holoenzyme.
Therefore,

Active site: The small specific area on the surface of the enzyme that binds the
substrate(s) and transforms is called active site.
OR
The three –dimensional entity containing amino acid residues from different parts
of the enzyme molecule is called active site.
Binding of the substance to the enzyme depends upon the precise arrangement of
atoms of both the active site and the substrate. Binding will occur by the
interaction between the functional groups of the substrate and those of the enzyme

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

and its cofactors. For proper interaction, the various functional groups have to be
precisely oriented and positioned.
Enzyme specificity: Enzymes are highly specific for the reaction they catalyse and
produce only the expected products from the given substrates. Enzyme specificity
greatly contributes towards the ordered metabolism that makes life possible. It not
only saves energy for cells, it also does not allow the potential build-up of toxic
by-products.
1) Group enzyme specificity: The enzymes transform substrates which have
one group in common is called group enzyme specificity.
Example: Hexokinase phosphorylates glucose, fructose and mannose
(hexose sugars)

i.e.

2) Absolute enzymes specificity: The enzymes act only one substrate is called
absolute enzyme specificity.
Example: a) Succinate dehydrogenase catalysis the oxidation of only
succinic acid

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

b) Glucokinase catalyses the phosphorylation of only glucose.

3) Stereospecificity enzymes specificity: The enzymes act on only one

stereoisomers (enatiomers) is called stereospecificity enzyme specificity.
For example: Lactate dehydrogenase oxidizes only L-lactate and not D-
lactate

Classification of enzymes: Enzymes are classified into following six

categories (OTHLIL)
1) Oxidoreductases: Enzymes which are catalyses oxidation and reduction
reactions are called oxidoredutases.

Example: Conversion of alcohols to aldehyde by using alcohol

dehydrogenase as enzyme

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

2) Transferases: Enzymes which are catalyses the transfer of a molecular

group from one molecule to another are called transferases.

Example: Transfer of a phosphate group from ATP to glucose by using

glucokinase as enzyme

3) Hydrolases: Enzymes which are catalyses the bond breakage by the

inducing the water are called hydrolases.

Example: Conversion of maltose to glucose by using maltase as enzyme

catalyst.

4) Lyases: Enzymes which are catalyses the reactions involving removal of

a group to form double bond or addition of group to a double bond are
called lyases

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

Example: Conversion of L-malate to fumarate using fumarase as enzyme

5) Isomerases: Enzymes which are catalyses the reactions involving

intramolecular rearrangements are called isomerases.

Example: Rearrangment of D-glyceraldehyde -3-phosphate to

dihydroxyacetone phosphate using triosephosphate isomerase as enzyme

6) Ligases: Enzymes which are catalyses the reaction by joining the two
molecules together are called ligases.

Example: Joining of bicarbonate and pyruvate molecules together to

form oxaloacetate by using pyruvate carboxylase as enzymes.

Enzyme –substrate interaction: Two theories have been proposed to

explain the interaction between enzymes and substrates.

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

a) Fischer’s lock and key model theory: According to this theory, the
active site of the enzyme is complementary in conformation to the
substrate, so that the correct substrate (key) fits into the active site of
the enzyme (lock). This theory emphasized stereospecificity of
catalysis.

b) Koshland’s induced –fit model theory: According to this theory, the

enzyme changes shape upon binding the substrate, so that the
conformation of substrate and enzyme protein are only
complementary after the binding reaction. Thus presumably brings the
amino acid side chains of the active site into positions of optimal
catalytic efficiency. This theory is more favoured because distortion in
the shapes of both enzyme and substrate has been observed on
binding.

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

Enzyme kinetics: Since enzymes are changes the rate of the reaction, so
following factors are affect on the rate of the reaction.
1) Enzyme concentrations: At the constant of pH, temperature and
concentration of substrate, the rate of the reaction is directly
proportional the concentration of enzymes. As the concentration of
enzymes increases, large number of substrate molecules can interact
with enzyme molecules and hence and hence rate of the reaction also
increase.
Rate of reaction

Enzyme concentration

2) Substrate concentration: At the constant of pH, temperature and

concentration of enzymes, the rate of the reaction initially increases
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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

very rapidly with substrate concentration at low substrate

concentration i.e., when the substrate concentration is low, the
reaction is first order with rate being proportional to [S]. Enzyme
active sites are still free and so available for catalysis. As substrate
concentration continues to increase, the increase in the rate of the
reaction begins to slow down i.e. at mid-[S], the reaction is mixed
order (i.e. proportionally changes). This happens because the number
of free enzyme active sites is reduced due to attachment of the
substrate molecules.
At a very high substrate concentration, no further increase in rate
is observed i.e. at high substrate concentration, the reaction is zero
order and rate is independent of [S]. This is because all available
active sites are saturated and so are working at full capacity.
The relationship of concentration of substrate to the rate of the
reaction is given by Michaelis – Mentenin the following graph and
equation

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

The equation is derived based on the assumption that the

enzyme reversibly combines with its substrate to form an ES complex
that subsequently breaks down to products regarding the free
enzyme

Characteristics of K m: The Michaelis –Mentin constant is

characteristic of an enzyme and particular substrate. It reflects the
affinity of the enzyme for the substrate. Km is numerically equal to
the substrate concentration at which the velocity of the reaction has
half the maximum value. Km does not vary with the concentration of
the enzyme.
Small Km: A numerically low value of Km signifies a high affinity of the
enzyme for the substrate. This means that a low concentration of the
substrate, the enzyme is half saturated and a velocity 1/2V max is
attained.

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

Large Km: A numerically high value of Km signifies a low affinity of the

enzyme for the substrate. This means that a high concentration of
the substrate is needed to half saturate the enzyme.
Evaluation of Km and Vmax: Accurate evaluation of Km and Vmax is not
possible from the hyperbolic curve of [s] vs V 0. However when 1/V0 is
plotted against 1/[S] a straight line is obtained. This plot is called
Lineweaver-Burke plot or the double reciprocal plot. Km and Vmax can
be evaluated from the graph as shown below.

3) Temperature: Most of the enzymes show an increase of 50% - 300%

in reaction rate when the temperature is increased by 10 0C. This trend

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

continues until about 450C. When denaturation of protein enzymes

sets in. from this point, the reaction rate drastically falls to zero.

Rate of the reaction

Temperature at which
denauration sets in

-----------------
Temperature 0C
4) pH (Hydrogen ion concentration): Most enzymes have a
characteristic optimum pH at which their activity is maximal. At this
pH, the catalytic groups in the active site are in the catalytically most
effective state of ionization. Above or below this Ph Z there is a
change from the required state of ionization and the rate is reduced. At
extreme pH values, the enzyme is inactivated due to denaturation.
Thus on either side of the optimal pH, reaction rate decreases sharply.
Rate of the reaction

Optimal pH
-------------------

pH

Allosteric enzyme: Allosteric enzymes are enzymes that have an additional

binding site for effector molecules other than the active site. The binding brings
about conformational changes, thereby changing its catalytic properties. The
effector molecule can be an inhibitor or activator.

Allostery is the process of enzyme regulation, where binding at one site influences
the binding at subsequent sites.

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

Examples:

1) Aspartate Transcarbamoylase (ATCase) catalyses the biosynthesis of

pyrimidine. Cytidine triphosphate (CTP) is the end product and also inhibits the
reaction. It is known as feedback regulation. ATP (adenosine triphosphate), a
purine nucleotide activates the process, high concentration of ATP can overcome
inhibition by CTP. This ensures the synthesis of pyrimidine nucleotide when a high
concentration of purine nucleotide is present

2) Glucokinase plays an important role in glucose homeostasis. It converts glucose

to glucose-6-phosphate and enhances glycogen synthesis in the liver. It also senses
the concentration of glucose for the release of insulin from pancreatic beta cells.
The glucokinase has low affinity for glucose, so it acts when more concentration of
glucose is present in the liver, which should be converted to glycogen. The activity
of glucokinase is regulated by glucokinase regulatory proteins

3) Acetyl-CoA Carboxylase regulates the process of lipogenesis. This enzyme is

activated by citrate and inhibited by a long chain acyl-CoA molecule such as
palmitoyl-CoA, which is an example of negative feedback inhibition by product.
Acetyl-CoA carboxylase is also regulated by phosphorylation/ dephosphorylation
controlled by hormones such as glucagon and epinephrine

Enzymes Metabolic pathway Inhibitor Activator

Helokinase Glycolysis Glucose-6 - -
phosphate
Phosphofructo Glycolysis ATP AMD, ADP
kinase

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

Isocitrate Krebs cycle ATP ADP, NAD

dehydrogenase
Acetyl CoA Fatty acid Palmitate Isocitrate
carboxylase synthesis

Enzyme inhibition: Many substances can reduce or eliminate the catalytic

activity of specific enzymes. These substances are called inhibitors. Inhibitors are
compounds that bind to an enzyme and prevent either the formation of the enzyme-
substrate complex or its breakdown to the enzyme and product(s)
Inhibitor may be irreversible or reversible. Irreversible inhibitors like
iodoacetate and the antibiotic penitilin usually bind covalently to one of the side
chain groups of the active site, thereby eliminating enzyme activity. Many nerve
gases and pesticides are irreversible inhibitors of vital enzymes of the nervous
system.
Reversible inhibitors may be easily removed by methods like dialysis from the
enzyme solutions.
Types of reversible inhibition: There are two common types of reversible
inhibition.
1) Competitive inhibition: In this inhibition, the structure of the competitive
inhibitor resembles the structure of the substrate of the enzyme i.e.
competitive inhibitors are substrate analogues. Therefore they compete with
the substrate for the active site, blocking it by not reacting.
Examples: Malonate competitively inhibits the enzyme succinate dehydrogenase
which converts succinate to fumarate.

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

Similarly, benzamide competes with arginine residues of peptides for the active
site of trypsin.
Sulpha drugs are another example due to their structural similarity with
paraaminobenzoic acid which is essential to vital bacterial enzymes. The effect of
competitively inhibitors can be reversed be increasing the substrate concentration.
People with methanol poisoning are treated by administrating ethanol.(here
methanol is a competitive inhibitor or alcohol dehydrogenase, whose substrate is
ethanol).

2) Non- competitive inhibition: In this inhibition, the inhibitor binds to the

enzyme at a site away from the active site. These are not substrate
analogues, and can bind either to the enzyme substrate complex. This
causes a change in the three dimensional conformation of the enzyme,
preventing the reaction from occurring.
Example: AMP noncompetitively inhibits the enzyme fructose-1,6-diphosphate
phosphatase, which converts fructose – 1,6-diphosphate to fructose -6-phosphate.

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC,K R PURAM,BENGALURU-36

Pepstatin noncompetitively inhibits the enzyme rennin. The effect of

noncompetitive inhibitors cannot be reversed by increasing substrate concentration.

Differences between competitive and non-competitive inhibitors

Competitive inhibitor
1) These are substrate analogues
2) Bind directly to the empty active
site, blocking it by not reacting
3) Effect can be reversed by increasing
substrate concentration
Non-competitive inhibitor
1) These are non-substrate analogues
2) Bind at a site other than the active
site, whenever the substrate occupies
it or not, and causes a conformational
change which prevents the reaction
from occurring.
3) Effect cannot be reversed by
increasing substrate concentration

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