ENZYMES AND COENZYMES-P1

BIOCHEMISTRY
BPS 2113

By Shashidharan Menon
 Historical Background
 Berzelius in 1836 coined the term catalysis (Greek: to dissolve). In 1878, Kuhne
used the word enzyme (Creek: in yeast) to indicate the catalysis taking place in

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the biological systems.

 lsolation of enzyme system from cell-free extract of yeast was achieved in 1883
by Buchner. He named the active principle as zymase (later found to contain a
mixture of enzymes), which could convert sugar to alcohol.

 ln 1926, James Sumner first achieved the isolation and

crystallization of the enzyme urease from jack bean and
identified.
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 Defination
 Enzymes are biocatalyst-s the catalyst of life. A catalyst is defined as a substance
that increases the velocity or rate of a chemical reaction without itself

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undergoing any change in the overall process.

 Enzymes may be defined as biocatalysts synthesized by living cells. They are

protein in nature (exception - RNA acting as ribozyme), colloidal and
thermolabile in character, and specific in their action.

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 Nomenculture and Classification
 The suffix-ase was added to the substrate naming the enzyme

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 Enzyme are sometimes consider under two broad categories :
 Intracellular enzymes -They are functional within cells where they are
synthesized.
 Extracellular enzymes – These enzymes are active outside the cell; all the
digestive enzymes belong to this group.

 The International Union of Biochemistry appointed an Enzyme Commission in

1961. This committee made a thorough study of the existing enzymes and
devised some basic principles for the classification and nomenclature 0f
enzymes.
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 Enzymes are divided into six major classes (in that order) based on type of
reaction catalysed by the enzyme.
 Nomenculture and Classification
 The oxidoreductases (class 1) catalyse the transfer of reducing equivalents
(hydrogen and electrons)from one redox system to another.

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 The transferases (class 2) catalyse the transfer of other groups from one
molecule to another. Oxidoreductases and rises generally require coenzymes

 The hydrolases (class 3) hydrolases cause cleavage of bond using water

 Lyases (class 4, often also referred to as"synthases") catalyse reactions involving

either the cleavage or formation of chemical bonds, with double bonds either
arising or disappearing. Cleavage of bond does not require water.

 The isomerases (class 5) move groups within a molecule, without changing the 5
gross composition of the substrate.
 Nomenculture and Classification
 The ligation reactions catalyzed by ligases ("synthetases," class 6) are energy-
dependent and are therefore always coupled the hydrolysis of nucleoside

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triphosphates

 Each enzyme is entered in the Enzyme Catalogue with a four-digit Enzyme

Commission number (EC number). The first digit indicates membership of one of
the six major classes. The next two indicate subclasses. The last digit indicates
where the enzyme belongs in the sub-subclass.

 For example, hexokinase is ATP:D-hexose 6-phosphotransferase E C. 2.7.1.1.

Identifies hexokinase as a member of class 2 (transferases), subclass 7 (transfer
of a phosphoryl group), sub-subclass 1 (alcohol is the phosphoryl acceptor), and
"hexose-6" indicates that the alcohol phosphorylated is on carbon six of a 6
hexose. However, it is still called as hexokinase.
 Nomenculture and Classification

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 Nomenculture and Classification

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 Units of Ezyme Activity
Katal

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 In order to maintain uniformity in the expression of enzyme activities (as units)
the Enzyme Commission of IUB has suggested new unit- namely katal (

 One kat denotes the conversion of one mole substrate per second (mol/sec).
Activity may also be expressed as millikatals (mkat), microkatals (µkat) and so on.

International Units (lU)

 One Sl unit or International Unit (lU) is defined as the amount of enzyme activity
that catalyses the conversion of one micromol of substrate per minute. Sl units
and katal are inter-convertible. 9
 Properties of Enzyme
Biological Catalyst which lowers the energy input/activation energy required for a
chemical reaction to proceed to speeds up rate of reaction.

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Enzymes are proteins

Enzymes remain unchanged after a reaction and therefore can work again.

Enzymes are specific to a substrate of a reaction (Lock and Key).

Enzymes are reversible and can catalyse a reaction going both ways (Synthesis/Lysis)

Enzymes are denatured by:

Change in Temperature
Change in pH
 Chemical Nature of Enzyme
 All the enzymes are invariably it has own tertiary structure and specific
conformation which is very essential for its catalytic activity.

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 The functional unit of the enzyme is known as holoenzyme which is often made
up of apoenzyme (the protein part) and a coenzyme (non-protein organic part).

Holoenzyme Apoenzyme + Coenzyme

(active enzyme) (protein) (non-protein)

 Term Prosthetic group is used when the non-protein moiety tightly (covalently)
binds with the apoenzyme. Coenzyme can be separated by dialysis from the
enzyme while the prosthetic group cannot be.
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 Monomeric enzyme is used if it is made up of a single polypeptide e.g.
ribonuclease trypsin.
 Chemical Nature of Enzyme
 Oligomeric enzymes which possess more than one polypeptide (subunit) chain
are known as e.g. lactate dehydrogenase, aspartate trans-carbamoylase

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 Multi-enzyme complexes possessing specific sites to catalyse Different reactions
in a sequence. Only the native Intact multi enzyme complex is functionally active
and not the individual units, if they are separated e.g. pyruvate dehydrogenase.

 Hybrid enzymes : lt is possible to rearrange genes and produce fusion proteins.

e.g. a hybrid enzyme (of glucanase and cellulase) that can more efficiently
hydrolyse barley B-glucans in beer manufacture.

 Site-directed mutagenesis : Technique used to produce a specified mutation at a

pre-determined position in a DNA molecule by incorporation of a desired amino 11
acid (of one's choice) in place of the specified amino acid in the enzyme.
 Factor Effecting Ezyme Activity
Concentration of enzyme

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 As the concentration of the enzyme is increased, the velocity of the reaction
proportionately increases .

 This property of enzyme is made use in determining

the serum enzymes for the diagnosis of diseases.

 By using a known volume of serum, and keeping all

the other factors (substrate, pH, temperature etc.) at
the optimum level, the enzyme could be assayed in
the laboratory.
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 Factor Effecting Enzyme Activity
Effect of temperature

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 Velocity of an enzyme reaction increases with increase in temperature up to a
maximum and then declines.

 A bell-shaped curve is usually observed.

 Increase in temperature results in higher activation

energy of the molecules and more molecular
(enzyme and substrate) collision and interaction for
the reaction to proceed faster.

 The optimum temperature for most of the enzymes 13

is between 40'C-45'C. However, a few enzymes (e.g.
venom phosphokinases are active even at 100'C.
 Factor Effecting Enzyme Activity
Effect of temperature

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 Temperature coefficient or Q10 is defined as increase in enzyme velocity when
the

 Temperature is increased by 10"C. For a majority of enzymes, Q10 is between

0"C and 40”C.

 Some plant enzymes like urease have optimum activity around 60'C. This may be
due to very stable structure and conformation of these enzymes.

 In general, when the enzymes are exposed to a temperature above 50"C,

denaturation leading to derangement the native (tertiary) structure of the 14
protein and active site are seen. Majority of the enzymes become inactive at
higher temperature (above 70'C).
 Factor Effecting Ezyme Activity
Effect of pH

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 lncrease in the hydrogen ion concentration (pH) considerably influences the
enzyme activity and a bell-shaped curve is normally obtained. Most of the
enzymes of higher organisms show optimum activity around neutral pH (6-8).
 Each enzyme has an optimum pH at which the
velocity is maximum. Below and above this pH, the
enzyme activity is much lower and at extreme pH,
the enzyme becomes totally inactive.

 Hydrogen ions influence the enzyme activity by

altering the ionic charges on the amino acids
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(particularly at the active site), substrate, ES complex
etc.
 Factor Effecting Ezyme Activity
Effect of product concentration

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 The accumulation of reaction products generally decreases the enzyme velocity.

 For certain enzymes, the products combine with the active site of enzyme and
form a loose complex and, thus, inhibit the enzyme activity.

 ln the living system, this type of inhibition is generally prevented by a quick

removal of products formed. The end product inhibition by feedback
mechanism.

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 Factor Effecting Ezyme Activity
Effect of activators

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 Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+,
Zn2+, Ca2+, Cu2+, Na+, K+ etc for their optimum activity. Rarely, anions are also
needed for enzyme activity e.g. chloride ion (C11 for amylase).

 Metals function as activators of enzyme velocity through various mechanisms

combining with the substrate, formation of ES-metal complex, direct
participation in reaction and bringing a conformational change in the enzyme.

 Metal-activated enzymes : The metal is not tightly held by the enzyme and can
be exchanged easily with other ions e.g. ATPase (Mg2* and Ca2*)
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 Metalloenzymes : These enzymes hold the metals rather tightly which are not
readily exchanged. e.g. alcohol dehydrogenase contain zinc.
 Factor Effecting Ezyme Activity
Effect of time

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 Under ideal and optimal conditions (like pH, temperature etc.), the time required
for an enzyme reaction is less. Variations in the time of the reaction are generally
related to the alterations in pH and temperature.

Effect of light and radiation

 Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain

enzymes due to the formation of peroxides. e.g. UV rays inhibit salivary amylase
activity.

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 Factor Effecting Ezyme Activity
Concentration of substrate

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 Increase in the substrate concentration gradually increases the velocity of
enzyme reaction within the limited range of substrate levels. A rectangular
hyperbola is obtained when velocity is plotted against the substrate
concentration

 Three distinct phases of the reaction are observed

 A-linear
 B-curve
 C-almost unchanged

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 Michaelis-Menten Equation
Enzyme kinetics and Km, value :

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 The enzyme (E) and substrate( S) combine with each other to form an unstable
enzyme-substrate complex (ES) for the formation of product (P).

 Here k1 , k2 and k3 represent the velocity constants for the respective reactions,
as indicated by arrows.

 The Michaelis-Menten Equation is a differential equation used to model the rate

at which enzymatic reactions occur. The Michaelis-Menton equation describes
how reaction velocity varies with substrate concentration 20
 Michaelis-Menten Equation
This model allows scientist to predict how fast a reaction will take place based on
the concentrations of the chemicals being reacted

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 Vmax = the maximum velocity achieved by the system, at maximum (saturating)
substrate concentrations.
 Km (the Michaelis constant) is the substrate concentration at which the reaction
velocity is 50% of the Vmax.
 [S] = concentration of the substrate S.

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 Michaelis-Menten Equation
In order to model an enzymatic reaction, some conditions must be maintained:

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 Temperature, ionic strength, pH, and other physical conditions that might affect
the rate must remain constant

 Each enzyme can act on only one other molecule at a time. The enzyme must
remain unchanged during the course of the reaction.

 The concentration of substrate must be much higher than the concentration of

enzyme. [ES] does not change with time (steady-state assumption). The rate of
[ES] formation is equal to that of the breakdown of ES (to E+S and to E+P)

 The rate of reaction is measured as soon as enzyme & substrate are mixed. At 22
that time, the [P] is very small, therefore, the rate of back reaction from P to S
can be ignored
 Michaelis-Menten Equation
 Meaning of Km (Michaelis constant) is numerically equal to the [S] at which the
reaction velocity is equal to ½Vmax. Does not vary with the concentration of

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enzyme

 Used to indicate how well a substrate interacts with an

enzyme (reflects affinity of the enzyme for that substrate)

 The smaller the value of Km, the tighter is the interaction

between substrate and enzyme E.g. A Km of 10-7M indicates
that the substrate has a great affinity for the enzyme than if
the Km is 10-5M

 Small Km : high affinity, a low [S] is needed to half saturated

the enzyme 23
 Large Km: low affinity, high [S] is needed to half-saturated
the enzyme
 Michaelis-Menten Equation
Turnover number (Kcat)

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 The number of molecules of substrate converted to product per unit time per
molecule of enzyme

 Unit of time-1

 The larger the value of Kcat for an enzyme, the faster the reaction will be

Relationship of velocity to enzyme concentration

 Rate of reaction α enzyme concentration

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 E.g. if the [E] is halved, the initial rate of reaction (Vo), as well as Vmax, are
reduced to half that of the original
 Michaelis-Menten Equation
Order of Reaction

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When [S] is much smaller than Km
 The velocity of the reaction is approx. proportional to the substrate
concentration The rate of reaction is then said to be first order with respect to
substrate

When [S] is much larger than Km

 the velocity is constant and equal to Vmax 100% of enzyme is in the ES complex
the rate of the reaction is independent of [S]

 The rate of reaction is said to be zero order with respect to [S]

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 Michaelis-Menten Equation
 Zero Order. The rate of a zero-order reaction is independent of the concentration
of the reactant(s). Zero-order kinetics are observed when an enzyme is saturated

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by reactants.

 First Order. The rate of a first-order reaction varies linearly on the concentration
of one reactant. First-order kinetics are observed when a protein folds and RNA
folds (assuming no association or aggregation).

 Second Order. The rate of a second-order reaction varies linearly with the square
of concentrations of one reactant (or on the product of the concentrations of
two reactants). Second order kinetics are observed for formation of double-
stranded DNA from two single-strands.
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 Michaelis-Menten Equation
If a plot of [A] vs t is a straight line, then the reaction is zero order.

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If a plot of ln[A] vs t is a straight line, then the reaction is 1st order.

If a plot of 1/ [A] vs t is a straight line, then the reaction is 2nd order

First Order Reaction Second Order Reaction

0.0E+00 2.0E+04 4.0E+04 6.0E+04

1/[A] (L /mol)
250
0
200
ln [A} (in mol / L

-1
150
-2
100
-3
50
-4
0
-5 0 500 1000
-6 27
Time (in seconds)
Time (in seconds)
Lineweaver-Burk plot

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Double reciprocal plot 1/vo versus 1/[s]

Used to calculate Km and Vmax

 X-intercept: -1/Km
 Y-intercept: 1/vmax
 Slope: Km/Vmax

Determine the mechanism of action of

enzyme inhibitors
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 Enzyme Active Site
 Enzymes are big in size compared to substrate which are relatively smaller

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 The active site (or active centre) of an enzyme represents as the small region at
which take substrate(s) binds and participates in the catalysis.

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 Enzyme Active Site
Characteristic of active site

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 The existence of active site is due to the tertiary structure of protein resulting in
three dimensional native conformation.

 The active site is made up of amino acids which are far from each other in the
linear sequence of amino acids (primary structure of protein).
Example
Enzyme lysozyme has 129 amino acids. The active site is formed by the contribution
of amino acid residues numbered 3 5, 52, 62, 63 and 101.

 Active sites are regarded as clefts or crevices or pockets occupying a small region
in a big enzyme molecule 30
 Enzyme Active Site
Characteristic of active site

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 The active site is not rigid in structure and shape. lt is flexible to promote the
specific substrate binding.

 Active site possesses a substrate binding side

and a catalytic site. The latter is for the
catalysis of the specific reaction.

 The coenzymes or cofactors some enzymes

depend presented a part of the catalytic site.

 The substrate binds at the active site by weak non-covalent bonds. 31

 Enzymes are specific in their function due to the existence of active sites.
 Enzyme Active Site
Characteristic of active site

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 The commonly found amino acids at the active sites are serine, aspartate,
histidine,cysteine, lysine, arginine, glutamate, tyrosine, serine is the most
frequently found.

 The substrates binds the enzyme (E) at the actives site o form enzyme-substrate
complex (ES) then product( P) is released after the catalysis and the enzyme is
available for reuse.

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 Enzyme Inhibition
Reversible Inhibition (Competitive inhibition)

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 The inhibitor (l) which closely resembles the real substrate(S) is regarded as a
substrate analogue. The inhibitor competes with substrate and binds at the
active site of the enzyme but does not undergo any catalysis.

 As long as the competitive inhibitor holds the active site, the enzyme is not
available for the substrate to bind.

 Concentration of the substrate and number of inhibitor and respective affinity

with the enzyme determines the degree of competitive inhibition. Inhibition
could be overcome by a high substrate concentration. the Km- value increases
whereas V-max remains unchanged 33
 Enzyme Inhibition
Reversible Inhibition (Competitive inhibition)

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Example

 Methanol is toxic to the body when it is converted to formaldehyde by the

enzyme alcohol dehydrogenase (ADH). Ethanol can compete with methanol for
ADH. Thus, ethanol can be used in the treatment of methanol poisoning.

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 Enzyme Inhibition
Reversible Inhibition (Competitive inhibition)

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Example

 Statins is a inhibitors of hydroxymethyglutaryl- CoA

reductase (HMG-CoA reductase). Inhibit the first
committed step in cholesterol biosynthesis.
Competitive inhibitors of HMG-CoA reductase are
cholesterol-lowering drugs (decrease rate of cellular
cholesterol biosynthesis).

 Structural analogs of the natural substrate for HMG-

CoA reductase e.g., Mevacor (lovastatin) and Zocor 35
(simvastatin), Lipitor (atorvastatin).
 Enzyme Inhibition
Reversible Inhibition (Non-Competitive inhibition)

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 Non-competitive inhibition : The inhibitor binds at a site other than the active
site on the enzyme surface. This binding impairs the enzyme function.

 The inhibitor has no structural resemblance with the substrate however, there
usually exists a strong affinity for the inhibitor to bind at the second site. In fact,
the inhibitor does not interfere with the enzyme-substrate binding.

 But the catalysis is prevented, possibly due to a distortion in the enzyme

conformation. non-competitive inhibition, the Km value is unchanged while
Vmax is lowered
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 Enzyme Inhibition
Reversible Inhibition (Non-Competitive inhibition)

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Example
 Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can non-competitively inhibit the
enzymes by binding with cysteine sulfhydryl groups. The general reaction for
Hg2+ is shown below. Heavy metals also lead to the formation of covalent bonds
with carboxyl groups and histidine, often resulting in irreversible inhibition.

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 Enzyme Inhibition
Irreversible Inhibition

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 The inhibitors bind covalently with the enzymes and inactivate them, which is
irreversible. These inhibitors are usually toxic poisonous substances.

 Disulfiram (Antabuse@i)s a drug used in the treatment of alcoholism. lt

irreversibly inhibits the enzyme aldehyde dehydrogenase. Alcohol addicts, when
treated with disulfiram become sick due to the accumulation of acetaldehyde,
leading to alcohol avoidance.

 (Note ; Alcohol is metabolized by two enzymes. lt is

first acted upon by alcohol dehydrogenase to yield
acetaldehyde. The enzyme aldehyde dehydrogenase 38
converts acetaldehyde to acetic acid.).
 Enzyme Inhibition

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 Enzyme Inhibition
Suicide inhibition

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 Suicide inhibition is a specialized form of irreversible inhibition.

 ln this case, the original inhibitor (the structural analogue/competitive inhibitor)

is converted to a more potent form by the same enzyme that ought to be
inhibited. The so formed inhibitor binds irreversibly with the enzyme. This is in
contrast to the original inhibitor which binds reversibly.

Example

Allopurinol (used in the treatment of gout) an inhibitor of xanthine oxidase, gets

converted to alloxanthine, a more effective inhibitor of this enzyme. 40
 Enzyme Inhibition
 Organophosphorous Pesticides: Diisopropylphosphofluoridate (DIPF) – Suicide
Inhibitors of Acetylcholinesterase

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 DIPF form stable phosphoesters with the active site serine of
acetylcholinesterase, the enzyme responsible for hydrolysis and inactivation of
acetylcholine at cholinergic synapses.
 Irreversible inhibition of the enzyme leads to
accumulation of acetylcholine at these synapses
and consequent neurologic impairment.

 Poisoning by pesticides that contain

organophosphate compounds produces a variety
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of symptoms, including nausea, blurred vision,
fatigue, muscle weakness and, potentially death
 Enzyme Inhibition
Allosteric inhibition
 Allosteric enzymes have two states: a low affinity state dubbed the “T” state and

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the high affinity “R” state. Inhibitors work by preferentially binding to the T state
of an allosteric enzyme, causing the enzyme to maintain this low affinity state.
Example
 ATP allosterically inhibits pyruvate kinase to
prevent increased formation of pyruvate, so
less ATP is eventually formed.
 Phosphofructokinase allosterically inhibited
by citrate, an intermediate of the Kreb’s
cycle.
 This means that glycolysis will be limited 43
when there is high ATP generation from the
Kreb’s cycle.
 Enzyme Specificity
Reaction specificity

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 The same substrate can undergo different types of reactions, each catalysed by a
separate enzyme and this is referred to as reaction specificity.

Example
 An amino acid can undergo transamination, oxidative deamination,
decarboxylation, racemization etc. The enzymes however, are different for each
of these reactions

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 Enzyme Specificity
Substrate specificity

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 The substrate specificity varies from enzyme to enzyme.

 Absolute substrate specificity : Certain enzymes act only on one substrate e.g.
urease cleaves urea to ammonia and carbon dioxide.

 Relative substrate specificity : Some enzymes act on structurally related

substances this, in turn, may be dependent on the specific group or a bond
present. Trypsin hydrolyses peptide linkage involving arginine or lysine.

 Broad specificity : Some enzymes act on closely related substrates which is

commonly known as broad substrate specificity, e.g. hexokinase acts on glucose, 46
fructose/mannose and glucosamine and not on galactose. Structural similarity
among the first four compounds makes them a common substrate.
 Coenzymes
 The non-protein, organic, Iow molecular weight and dialyzable substance
associated with enzyme function is known as coenzyme.

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 Coenzymes are second substrates :

 Coenzymes are often regarded as the second substrates or co-substrates,

since they have affinity with the enzyme comparable with that of the
substrates

 Coenzymes undergo alterations during the enzymatic reactions, which are

later regenerated. This is in contrast to the substrate which is converted to
the product
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 Coenzymes
Coenzymes from B-complex vitamins :

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 Most of the coenzymes are the derivatives of water soluble B-complex vitamins.
In fact, the biochemical functions of B-complex vitamins are exerted through
their respective coenzymes.

Non-vitamin coenzymes :

 Not all coenzymes are vitamin derivatives. There are some other organic
substances which have no relation with vitamins but function as coenzymes.
They may be considered as non-vitamin coenzymes e.g. ATP, CDP, UDP etc. The

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 Coenzymes
Nucleotide coenzymes :

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 Some of the coenzymes possess nitrogenous base, sugar and phosphate. Such
coenzymes are, therefore, regarded as nucleotides e.g. NAD+, NADP+, FMN, FAD,
coenzyme A, UDPC etc.

Coenzymes do not decide enzyme specificity :

 A particular coenzyme may participate in catalytic reactions along with different

enzymes. For instance, NAD+ acts as a coenzyme for lactate dehydrogenase and
alcohol dehydrogenase. In both the enzymatic reactions, NAD+ is involved in
hydrogen transfer.
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 The specificity of the enzyme is mostly dependent on the apoenzyme and not on
the coenzymes.
 Mechanism of Enzyme Action
Enzymes lower activation energy

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 The energy required by the reactants to undergo the reaction is known as
activation energy. The reactants when heated attain the activation energy.

 The catalyst (or the enzyme in the biological system) reduces the activation
energy and this causes the reaction to proceed at a lower temperature.

 Enzymes do not alter the equilibrium constants, t hey only enhance the velocity
of the reaction.

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Mechanism of Enzyme Action

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Effect of enzyme on activation
energy if a reaction carried out
(A is the substrate) and B is the
product)

Enzyme decrease activation

energy

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 Mechanism of Enzyme Action
Enzyme substrate complex formation

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 The prime requisite for enzyme catalysis is that the substrate (S) must combine
with the enzyme (E) at the active site to form enzyme-substrate complex (ES)
and then enzyme-product complex (EPC) which ultimately results in the product
formation (P).

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 Mechanism of Enzyme Action
Lock and key model or Fischer's template theory

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 According to this model, the structure or conformation of the enzyme is rigid.
The substrate fits to the binding site (now active site) just as a key fits into the
proper lock or a hand into the proper glove.

 Thus the active site of an enzyme is a rigid and pre-shaped template where only
a specific substrate can bind. This model does not give any scope for the flexible
nature of enzymes, hence the model totally fails to explain the effect of allosteric
modulators.

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 Mechanism of Enzyme Action
Induced fit theory or Koshland's model

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 Koshland, in 1958, proposed a more acceptable and realistic model for enzyme
substrate-complex formation. As per this model, the active site is not rigid and
pre-shaped.

 The essential features of the substrate binding site are present at the nascent
active site. The interaction of the substrate with the enzyme induces a fit or a
conformation change in the enzyme, resulting in the formation of a strong
substrate binding site.

 Due to induced fit, the appropriate amino acids of the enzyme are repositioned
to form the active site and bring about the catalysis 54
 Mechanism of Enzyme Action
Induced fit theory or Koshland's model

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 Induced fit model has sufficient experimental evidence from the X-ray diffraction
studies. Koshland's model also explains the action of allosteric modulators and
competitive inhibition on enzymes.

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 Mechanism of Enzyme Action
Substrate strain theory

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 In this model, the substrate is strained due to the induced conformation change
in the enzyme.

 When a substrate binds to the preformed active site, the enzyme induces a
strain to the substrate. The strained substrate leads to the formation of
product.Combination of the induced fit model with the substrates train is
considered to be operative in the enzymatic action.

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 Mechanism of Enzyme Catalyst
Substrate strain :

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 lnduction of a strain on the substrate for ES formation is discussed above. During
the course of strain induction, the energy level of the substrate is raised, leading
to a transition state.

 The mechanism of lysozyme (an enzyme of tears, that cleaves p-1,4 glycosidic
bonds) action is believed to be due to a combination of substrates train and acid-
base catalysis.

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 Thermodinamic of Enzymatic Reaction
lsothermic reactions :
 The energy exchange between reactants and products is negligible. Glycogen

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phosphorylase
Glycogen + Pi →Glucose 1-phosphate

Exothermic (exergonic) reactions :

 Energy is liberated in these reactions. Urease
Urea → NH3 + CO2 + energy

Endothermic (endergonic) reactions :

 Energy is consumed in these reactions. Glucokinase
Glucose+ ATP → Glucose-6 -phosphate+ ADP
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 Regulation of Enzyme Activity
❖ In biological system, regulation of enzyme activities occurs at different stages

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in one or more of the following ways to achieve cellular economy.
➔ Allosteric regulation
➔ Activation of latent enzymes
➔ Compartmentation of metabolic pathways
➔ Control of enzyme synthesis
➔ Enzyme degradation
➔ lsoenzymes

2
 Isoenzyme
❖ Multiple forms of the same enzyme will also help in the regulation of enzyme
activity,Many of the isoenzymes are tissue-specific.Although isoenzymes of a

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given enzyme catalyses the same reaction, they differ in Km Vmax or both. e.g.
isoenzymes LDH and CPK

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 CLINICAL ENZYMOLOGY
❖ Plasma contains many functional enzymes, which are actively secreted into
plasma. For example, enzymes of blood coagulation.

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❖ On the other hand, there are a few non- functional enzymes in plasma, which
are coming out from cells of various tissues due to normal wear and tear. Their
normal levels in blood are very low; but are drastically increased during cell
death (necrosis) or disease. Therefore, assays of these enzymes are very useful
in diagnosis of diseases.

❖ The reference ranges for enzymes in plasma depends on the method of assay
used; and therefore will vary from laboratory to laboratory. Hence, the values
given in this book are only for a general guidance, and should not be taken as
absolute. 15
 Cardiac Biomarkers
❖ A biomarker is a clinical laboratory test which is useful in detecting dysfunction of
an organ. Cardiac biomarkers used to detect cardiac diseases, which may be:

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A. Acute coronary syndrome resulting from myocardial ischemia
B. Congestive cardiac failure due to ventricular dysfunction

❖ The different markers are used to:

I. Detect myocardial ischemia at the earliest
II. Montoring the progression of the condition
III. Predict the risk in cardiac dysfunction.

❖ Commonly used biomarkers early detection of acute myocardial infarction are:

I. Cardiac troponins, TnI and TnT
II. Creatine kinase, CK-MB 16
III. Myoglobin. Of these, troponins and CK-MB are the sensitive and specific
markers, whereas myoglobin though sensitive, is non-specific
 Cardiac Biomarkers
❖ Predictors of risk in cardiac disease are of two types:
I. Predicting the onset of ischemia

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II. Quantify the ventricular damage

❖ The risk predictors mainly include the atherogenic lipoproteins in plasma along
with the inflammatory marker like hsCRP (high sensitive C reactive protein).

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 Creatine Kinase (CK)
Reference Values

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❖ It catalyzes the reaction shown in Figure 23.1. It was called as creatine
phosphokinase in old literature. Normal serum value for CK is 15-100 U/L for
males and 10-80 U/L for females.

CK and Heart Attack

❖ CK value in serum is increased in myocardial infarction. The time course is

shown below. The CK level starts to rise within 3-6 hours of infarction.

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 Creatine Kinase (CK)
CK and Heart Attack

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❖ Therefore, CK estimation is very useful to detect early cases, where ECG
changes may be ambiguous. A second peak may indicate another ischemic
episode.

❖ The CK level is not increased in hemolysis or in congestive cardiac failure; and

therefore CK has an advantage over LDH. The area under the peak and slope of
initial rise are proportional to the size of infarct.

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 Creatine Kinase (CK)
Iso-enzymes of CK

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❖ CK is a dimer; each subunit has a molecular weight of 40 kD. The subunits are
called B or brain and M for muscle. Therefore, three iso-enzymes are seen in
circulation.

❖ Normally CK2 (heart iso-enzyme) is only 5% of the total activity. Even doubling
of the value of CK2 (MB) iso-enzyme may not be detected, if total value of CK
alone is estimated. Hence, the estimation of MB- isoenzyme is the best
diagnostic marker in myocardial infarction.

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 Creatine Kinase (CK)
CK and Muscle Diseases

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❖ The level of CK in serum is very much elevated in muscular dystrophies (500-
1500 IU/L),In female carriers of X-linked muscular dystrophy (heterozygous),
CK is moderately raised.

❖ CK level is highly elevated in crush injury, fracture and acute cerebrovascular

accidents.Estimation of total CK is employed in muscular dystrophies and MB
isoenzyme is estimated in myocardial infarction.

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 Cardiac Troponins
❖ They are not enzymes. However, Troponins are now accepted as reliable
markers for myocardial infarction, and hence discussed here.

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❖ Measurement of cardiac troponins have become one of the main tests in early
detection of an ischemic episode and in monitoring the patient.

❖ The troponin complex consists of 3 compo- nents; troponin C (calcium binding

subunit), troponin I (actomyosin ATPase inhibitory subunit), and troponin T
(tropomyosin binding subunit).

❖ Troponin I (TnI) is encoded by 3 different genes, giving rise to 3 isoforms; the

"slow" and "fast" moving forms are skeletal variety. Cardiac isoform is specific 22
for cardiac muscle; the amino acid sequence is different in skeletal muscle
isoform.
 Cardiac Troponins
❖ Cardiac isoform of CTnT and CTnI are mainly (95%) located in myofibrils and

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the remaining 5% is cytoplasmic. Circulating isoforms may be ternary
complexes (TIC) or binary complexes (IC) or free subunits of I and T. They are
generally identified and quantitated by immunological (ELISA or
immunoturbidimetric) reactions.

❖ Troponin I is released into the blood within 4 hours after the onset of
symptoms of myocardial ischemia; peaks at 14-24 hours and remains elevated
for 3-5 days post-infarction. Therefore, CTI is very useful as a marker at any
time interval after the heart attack. It is not increased in muscle injury;
whereas CK2 may be elevated in some muscle injury. The initial increase is due 23
to liberation of the cytoplasmic fraction and sustained elevation is due to the
release from myofibrils.
 Cardiac Troponins
❖ Serum level of Troponin T (TnT) increases within 6 hrs of myocardial infarction,

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peaks at 72 hours and then remains elevated up to 7-14 days.

❖ Cardiac troponin elevations at lower concentrations than the 99th percentile

value used for MI diagnosis may identify patients who have not had an MI but
still have a risk of having an adverse cardiac event.

24
 Lactate Dehydrogenase (LDH)
Reference Values

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❖ LDH will convert pyruvate to lactate (Chapter 9). Normal value of LDH in serum
is 100-200 U/L. Values in the upper range are generally seen in children.
Strenuous exercise will slightly increase the value. LDH level is 100 times more
inside the RBC than in plasma, and therefore minor amount of hemolysis will
result in a false positive test.

LDH and Heart Attack

❖ In myocardial infarction, total LDH activity is increased, while H4 iso-enzyme is

increased 5-10 times more. The time course of LDH level after a heart attack is
given in Figure 23.1. The magnitude of the peak value as well as the area 25
under the graph will be roughly proportional to the size of the myocardial
infarct.
 Lactate Dehydrogenase (LDH)
Differential Diagnosis

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❖ Increase in total LDH level is seen in hemolytic anemias, hepatocellular
damage, muscular dystrophy, carcinomas, leukemias, and any condition which
causes necrosis of body cells. Since total LDH is increased in many conditions,
the study of iso- enzymes of LDH is of more significance.

Iso-enzymes of LDH

❖ LDH enzyme is a tetramer with 4 subunits. But the subunit may be either H
(heart) or M (muscle) polypeptide chains. Although both of them have the
same molecular weight (32 kD), there are minor amino acid variations.
26
 Lactate Dehydrogenase (LDH)
Iso-enzymes of LDH

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❖ So 5 combinations of H and M chains are possible; H4, H3M, H2M2, M3H and
M4 varieties, forming 5 iso-enzymes. All these 5 forms are seen in all persons.
The iso- enzymes are usually separated by cellulose acetate electrophoresis at
pH 8.6 .

❖ M4 form is seen in skeletal muscles while H4 form is seen in heart.

❖ Normally LDH-2 (H3M1) concentration in blood is greater than LDH-1 (H4); but
this pattern is reversed in myocardial infarction; this is called flipped pattern.
27
LDH has only limited diagnostic value because of its non- specific nature.
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