ENZYMES:
DEFINATION:
Enzymes, are the catalysts of biological system that enhance
the rate of a biochemical reaction by lowering the activation energy,
and are molecular device that determines the pattern of chemical
transformation. They also mediate the transformation of one form
of energy into another.
• Characteristics of Enzymes:
1. Almost all enzymes are proteins and follow the physical and
chemical reactions of proteins.
2. They are heat labile
3. Are water soluble
4. Can be precipitated by protein precipitating reagents
5. Contain 16% wt as nitrogen
CLASSIFICATION OF ENZYMES
1. CLASSICAL NOMENCLATURE
• In early days, whimsical names were given, for e.g. Pepsin, trypsin,
chymotrypsin which are still used.
• Later, it was agreed to call the enzymes by adding suffix "-ase to the
substrate, eg. Enzyme Lactase acts on the substrate lactose. These are
called as trival names.
• Later, to avoid the ambiguity due to their trival names IUBMB system
of classification was introduced
2. IUBMB SYSTEM OF CLASSIFICATION:
International Union of Biochemistry and Molecular Biology suggested IUBMB system of
nomenclature of enzymes. It is complex but unambiguous.
As per this system, the name starts with EC enzyme commission number followed by 4 digits.
1st digit represents the class
2nd –the subclass
3rd –sub-sub class or sub group and
4th –number of the particular enzyme in the list
The enzymes are grouped into 6 major classes
Class 1: oxidoreductases
Class 2: transferases
Class 3: hydrolases
Class 4: lyases
Class 5: isomerases
Class 6: ligases
OXIDOREDUCATASES
• Catalyse redox reaction
• Transfer of hydrogen or addition of oxygen
Eg. Lactate dehydrogenase
Pyruvate + NAD------------------ lactate + NADH + H+
Alcohol +NAD --------------------aldehyde + NADH + H+
(trivial name) Alcohol dehydrogenase:
IUBMB name Alcohol-NAD-oxidoreductase. Code no. EC.1.1.1.1
TRANSFERASES
Transfer one group other than hydrogen from the substrate to another substrate.
Eg. Hexokinase
Glucose + ATP -------------------- Glucose-6-phosphate + ADP
HYDROLASES
. This class of enzymes can hydrolyse ester, ether, peptide or glycosidic bonds by
adding water and then breaking the bond
Eg. Acetyl choline + H2O ------------------ choline + acetate
Acetyl choline esterase
. All digestive enzymes
LYASES
• These enymes can remove groups from substrate or break bonds by
mechanisms other than hydrolysis ( adding water)
Eg. Aldolase
Fructose 1,6-bisphosphate --------------- glyceraldehyde 3 phosphate +
dihydroxyacetone phosphate
ISOMERASES
• They can produce optical, geometric or positional isomers of substrates.
Include racemases, epimerases, cis-trans isomerases
Glucose 6 phosphate -------------- fructose 6 phosphate ; phosphor-hexo
isomerase
LIGASES:
These enzymes link or condense two substrates
together, usually with the simultaneous hydrolysis
of ATP
Eg.acetyl Co carboxylase
Malony CoA synthetase
Acetyl CoA + CO2----------- Malonyl CoA
IMPORTANT TERMS:
ZYMOGEN OR PROENZYME:
• A number of proteolytic enzymes found in the blood or in the
digestive tract are present in an inactive form, called zymogen or
proenzymes, which must be cleaved to be activated
• Their synthesis in this form prevents them from catalyzing reactions in
the cell where they are synthesized
• Eg. Chymotrypsin is secreted by the pancreas as chymotropsinogen. It
is activated in the digestive tract by the proteolytic enzyme trypsin
COFACTORS, COENZYMES, ACTIVATORS, APOENZYMES..
Enzymes may be simple proteins or complex enzymes containing a non-protein
part, called the prosthetic group. They are also referred as co-enzymes, which are
organic compounds. If inorganic ions- they are called as activators.
The protein part of the enzyme is called as apo-enzyme.
These two portions combined together is called the holo-enzyme.
Co-factor is used as a collective term to include co-enzymes and metal ions
MECHANISM OF ENZYME ACTION
Formation of an enzyme-substrate (ES) complex is the first step in enzymatic
catalysis
Substrate is bound through multiple non-covalent interactions at the active site
of the enzyme forming an enzyme-substrate complex which is subsequently
converted to product and free enzyme.
E + S ========= ES -------------- E + P
Two models for substrate binding to the active site of the enzyme have been
proposed to explain the specificity that an enzyme has for its substrate:
1. Lock and key model or rigid template model of Emil Fisher
2. Induced fit model or hand in glove model of Daniel E Koshland
LOCK AND KEY MODEL
• Proposed by Emil Fisher
• In this model, enzyme is pre-shaped and the active site has a rigid structure
that is complementary to that of the substrate.
• Called as lock and key model because in this model the substrate fits into the
active site in much the same way that a key fits into a lock
• Useful in understanding how some enzymes can bind only a specific substrate
but will not bind another substrate with an almost identical structure. For eg.
Most enzymes in carbohydrate metabolism can bind to D-isomers but not L-
isomers
Limitations:
• Explains all mechanism but do not explain the changes in the enzymatic
activity in the presence of allosteric modulators.
• Explains the specificity of enzyme substrate interaction but the implied
rigidity of the enzymes active site failed to explain the dynamic changes that
HAND IN GLOVE MODEL
• More improvised and proposed by Daniel E Koshland
• Postulated that the enzymes are flexible and shapes of the active site can be
modified by the binding of the substrate.
• In this model, the substrate induces a conformational change in the enzyme such
that precise orientation of catalytic groups is effected, in the same manner in
which placing a hand (substrate) into a glove (enzyme) induces changes in the
glove's shape. Therefore known as hand in glove model.
• Allosteric inhibition can also be explained by this hypothesis of Koshland.
SPECIFICITY OF ENZYME
Following types of specificity have been recognized
1. Absolute specificity
2. Group specificity
3. Reaction specificity
4. Stereospecificity
ABSOLUTE SPECIFICITY:
• Certain enzymes will act on only one specific substrate and catalyse one reaction
Eg. Lactose ------------- glucose + galactose enzyme: lactase
Urea ---------------- ammonia + CO2 enzyme : urease
GROUP SPECIFICITY:
Some enzymes catalyse the same reaction on a group of structurally similar compounds
Eg. Hexokinase can catalyse phosphorylation of glucose, galactose and mannose
REACTION SPECIFIC:
Enzymes are specific in their action. Almost only one enzyme catalyzes a given specific
reaction
Eg. Pyruvate can undergo several reactions but all are carried by different specific enzymes
Pyruvate-------------acetyl CoA ; by PDH
Pyruvate ------------lactate ; by LDH
Pyruvate-----------alanine ; by transaminase
STEREO SPECIFICITY:
Many enzymes show specificity towards stereoisomers, i.e. they act on only one type of
isomer
Eg. Human enzymes are specific for L –amino acids and D- carbohydrates.
ENZYME KINETICS
The study of reaction rates and how they change in response to changes in
experimental parameters is known as kinetics
One of the key factors affecting the rate of a chemical reaction
catalyzed by an enzyme is the amount of substrate present. The
affect on initial velocity of varying substrate is shown in the figure.
Here k1, k2 and k3 represent the velocity constants for the respective
reactions, as indicated by arrows.
Here k1, k2 and k3 represent the velocity constants for the respective
reactions, as indicated by arrows.
Km, the Michaelis-Menten constant (or Brigrsand Haldane's constant), is
given by the formula
Here k1, k2 and k3 represent the velocity constants for the respective
reactions, as indicated by arrows.
Km, the Michaelis-Menten constant (or Brigrsand Haldane's constant), is given
by the formula
Km or the Michaelis-Menten constant is defined as the substrate concentration
(expressed in moles/l) to produce half-maximum velocity in an enzyme catalyzed
reaction. It indicates that half of the enzyme molecules (i.e. 50%) are bound with
the substrate molecules when the substrate concentration equals the K value.
FACTORS AFFECTING THE VELOCITY OF ENZYME REACTION/ ENZYME ACTIVITY
Enzyme activity is measured in terms of number of substrate molecules converted into
product by an enzyme molecule in a unit time. Various factors that affect enzyme
activity are
1.Substrate concentration
2. Enzyme concentration
3. pH i.e. H+ ion concentration
4. temperature
5. product concentration
6. enzyme activators
7. enzyme inhibitors
SUBSTRATE CONCENTRATION:
• For a given quantity of enzyme, the velocity of the reaction increases as the
concentration of the substrate is increased.
• At first, this relationship is almost linear, i.e. the velocity is directly proportional to
the substrate concentration and follows first order kinetics
• But later, the reaction curve becomes hyperbolic in shape, i.e. the activity remains
constant because there are no more enzyme molecules to act, as all the active sites
are occupied by substrate and a plateau is obtained. The reaction is said to exhibit
zero order kinetics.
Fig
ENZYME CONCENTRATION:
• The velocity of a reaction is directly proportional to the amount of enzyme present
• The substrate must be present at a concentration sufficient to ensure that all of the
enzyme have substrate bound to their active site
Fig.
pH:
• Each enzyme has a pH at which its activity is maximum called as optimum pH
• Below or above this pH, enzyme activity is decreased
• The optimum pH differs from enzyme to enzyme
• Eg. Optimum pH for pepsin = 1.2
For trypsin = 8.0
• A bell shaped curve is obtained when enzyme activity is plotted against the pH
• pH alters
1. Ionization state of the amino acids present in the active site of the
enzyme
2. Ionization state of the substrate
3. May dissociate apo-enzyme from the prosthetic group
4. Denaturation of the enzyme
TEMPERATURE:
• each enzyme shows the highest activity at a particular temperature called
optimum temperature
• the activity progressively declines both above and below this temperature
• increase in velocity is due to the increase in the kinetic energy to pass over the
energy barrier and form the product of the reaction
• further elevation of the temperature results in a decrease in reaction velocity as a
result of temperature induced denaturation of the enzyme.
• Low temperature also decreases enzyme activity and may be completely inactive
at temperatue of 0° C and below
• Eg. Most of the body enzymes have the optimum temperature close to 37-38°C
and have less activity as the temperature rises.
• Bell shaped curve
EFFECT OF PRODUCT
• Accumulation of products causes the inhibition of enzyme activity
• Reason for this is may be because of less availability of acitive sites
Eg. Glucose-6 phosphate accumulation inhibits the enzyme hexokinase
Glucose------------glucose -6- phosphate
ENZYME ACTIVATORS
• In presence of certain inorganic ions, some enzymes show higher activity
• E.g. chloride ions activate salivary amylase and calcium ions activates lipases
• Some enzymes needs to undergo cleavage for activation, e.g. trypsinogen cleaved
to become active trypsin
ENZYME INHIBITORS
Inhibitors will result in decrease in enzyme activity. This may be reversible and irreversible.
1.Reversible inhibitors
a) Competitive inhibition
Inhibitor compete with the substrate for the active site of the enzyme . The inhibitor must posses structure similarity
with the natural substrate to act as competitive enzyme activators.
Eg. Sulphonamides: bacteria needs folic acid and synthesize by using PABA(para aminobenzoic acid). Sulpha drug are
analogues of PABA and hence inhibit folic acid synthesis, and bacteria die. So, these drugs are used as antibacterial agents
b) Non competitive inhibition
There is no competition between the inhibitor and substrate for the active site . these are not influenced by concentration
of substrate . It inhibits by binding irreversibly to the enzyme but not at the active site . Which changes the shape of enzyme
and active site.
Inhibitors bind at sites other than substrate binding site
Eg. Cyanide inhibits cytochrome oxidase
c) Uncompetitive inhibition
Inhibitors bind to enzyme-substrate complex, but not to the free enzyme. Uncompititive inhibitors
frequently obserbed in multi substrate reaction.Inhibition can not be reversed by increasing the
substrate since inhibitor doesnot compete with substrate for the same binding site.
Eg. Inhibition of placental alkaline phosphatase by phenylalanine
2.Irreversible inhibition
a) Suicide inhibition
The inhibitor makes use of the enzyme's own reaction mechanism to inactivate it.
Eg. Aspirin inactivates an enzyme cyclooxygenase which catalyses the first reaction in the biosynthesis of
prostaglandins from arachidonic acid. Therefore aspirin is used as anti-inflammatory
Application of enzymes:
• Therapeutical uses
• Bacterial asparaginase for treatment of leukemia
• Lysozyme antibacterial action is abundant in secretion of tears, saliva, human milk mucus etc.
• Analytical uses
• Glucose estimation in blood…GOD POD(glucose oxidinase and peroxidase)
• Genetic engineering
• Taq DNA polymerase used in PCR(Polymerase chain reaction)
• Industrial application
• Protease in washing powder and leather as well as textile industries
• Alpha amylase used to produce glucose from starch
• The wine and fruit juice is usually achieved by treatment with fungal pectinases .
• Diagnostic uses
• Diagnostic enzymes- iso enzymes ie . Lactate dehydrogenase used to catalyze the synthesis
of glucose in heart and skeletal muscle. And serum acid phosphatase will be elevated in the
body of patient with prostate cancer.
