ENZYMES
ENZYMES: BASICS
 Rate of a Reaction
 Reversible & Irreversible Reactions
 Reaction Equilibrium
 Catalysis
BACKGROUND: A REACTION
r1
A+B
 Reaction rate:
r1 α [A] [B]
 Therefore,
r1 = k1 [A] [B]
C+D
REVERSIBLE REACTION
r1 (k1)
A+B
C+D
r2(k2)
 forward reaction (left to right)
 backward reaction (right to left)
 state of equilibrium – chemical equilibrium.
 At equilibrium,
r1 = r2
BACKGROUND
 r1 = k1 [A] [B]
&
r2 = k2 [C] [D]
 At equilibrium,
k1 [A] [B] = k2 [C] [D]
[A] [B]
[C] [D]
k2 = Keq(equilibrium constant)
=
k1
 Law of mass action - for reversible reactions
(Effects of [S]and [P] on direction in net reaction proceeds)
BACKGROUND
K eq=
[A] [B]
[C] [D]
 Freely reversible reaction,
K eq value is 1
There is no energy change (ΔG = 0);
 Irreversible reaction
K eq value
very high (endergonic reaction; ΔG = +ve )
or
Negligible(exergonic reaction; ΔG =–ve).
CATALYSIS
 Catalyst increases rate of a chemical reaction , remains unchanged
chemically at the end of the reaction
 Phenomenon is CATALYSIS
 Neither cause chemical reactions to take place
 Nor change the equilibrium constant of chemical reactions
 Reactants bind to catalyst, products are released
CATALYST
 Catalyze forward & backward reactions equally
 Only catalyze reaction in thermodynamically allowed direction
 Catalysts accelerate chemical reactions by lowering the activation
energy or energy barrier
EFFECT OF CATALYST ON
ACTIVATION ENERGY OF A REACTION
ENZYMES: DEFINITION
Biocatalysts synthesized by living tissues
which increase the rate of reaction without
getting consumed in the process
ENZYMES
 Biocatalysts- Neither consumed / permanently altered
 Colloidal organic compounds – proteins
 Formed by living organisms
 High specificity for their substrates and reaction types
 No formation of unnecessary by-products
 Function in dilute aqueous solutions under mild conditions of
temperature and pH
 Subjected to physiological regulation
 Several enzymes can work together in a specific order creating metabolic pathways
ENZYMES: MEDICAL IMPORTANCE
 Accelerate 106 to 1012 times
 Regulatory enzymes sense metabolic signals
 Inherited genetic disorders(Phenylketonuria)
 Inhibitors of enzymes can be used as drug
Lovastatin for HMG CoA reductase
Allopurinol inhibits Xanthine oxidase – treatment of gout
 Clinical enzymology
ENZYMES: HISTORY
 En-zyme = in yeast
 In 1850s Louis Pasteur
“ferments”
fermentation of sugar into alcohol by yeast
 Urease – first enzyme to be isolated in crystalline form in 1926
 Ribozymes
 Made up of RNA
SPECIFIC CATALYSIS
 Specific for type of reaction & single substrate or related substrates
 Stereospecific catalysts
 D-sugars and L-amino acids
 “ three point attachment”
 Nonchiral substrates to chiral products; eg. Pyruvate to L-lactate
ACTIVE SITE
 Size of enzymes >substrates
 “small region at which the substrate binds and catalysis take place”
 Imparts efficiency:
Local concentration
Shields substrate from solvent
 Situated in a pocket or cleft of the enzyme
 The active site contains
Substrate binding site (substrate)
Catalytic site (reaction)
ACTIVE SITE
 Catalytic sites contain sites for binding cofactors or coenzymes
 d/t tertiary structure of protein
 loss of native enzyme structure  derangement of active site loss
of function
 Catalysis  Substrate/s should bind to substrate binding site
reversibly by weak non-covalent bond
(Hydrogen bond, Van der walls force, hydrophobic interactions)
ACTIVE SITE
 Not rigid in structure and shape
 Flexible to promote effective binding of substrate to the enzyme
 Responsible for substrate specificity
 If an enzyme is denatured or dissociated into subunits  catalytic
activity is lost
 Enzymes acts within the moderate pH and temperature
ACTIVE SITE
 In active site 3–4 amino acids directly involved in catalysis- catalytic
residues
 Amino acids far away in the primary structure contribute to formation
of active site
 amino acids at the active sites –
*serine
*aspartate
*histidine
*cysteine
*Lysine
*arginine
*glutamate
*tyrosine
ACTIVE SITE
ENZYMES AND
ENZYME CATALYZED REACTIONS
 Substrate: Reactant/s on which enzymes act to catalyze the reaction
 Enzymes are much larger than the substrates they act on substrates
on which they act
HOLOENZYME, APOENZYME
 Some enzymes contain a non protein prosthetic group other than
protein component
 Holoenzyme=Apoenzyme+cofactor
(protein) (non protein)
CO FACTORS
 Non-protein factors required for catalysis
 Bind to catalytic site
Organic cofactors
Prosthetic groups  tightly bound
coenzymes – released from active site during reaction
Inorganic cofactors
Activators
 Exceptions: FMN, FAD & biotin are tightly bound to enzymes
 But called as coenzymes
CO FACTORS
coenzymes
organic
Prosthetic
group
cofactors
inorganic
activators
PROSTHETIC GROUP
 Tightly bound to enzyme by covalent bond
 cannot be separated from enzyme by Dialysis
 Biotin in carboxylases
 Apoenzyme + Prosthetic group = Holoenzyme
(active enzyme)
CO ENZYMES
 Derivatives of vitamins
 Bound reversibly by weak non-covalent bonds to active site
 Released during reaction
 Separated easily from enzyme by dialysis
 Affinity for the enzyme is similar to substrate
 chemically changed by catalysis
 Altered in reaction & regenerated to original structure in subsequent
reaction Co-substrate
 Function: carriers of various groups
CO ENZYMES
 Hydrolases (class 3) - not require coenzymes
 IUB classes: I,II,V and VI need coenzymes
Coenzyme form
Vitamin
(derived from)
Group transferred
Thiamine pyrophosphate (TPP)
Thiamine(Vit B1)
Hydroxy ethyl
FAD and FMN
Riboflavin (Vit B2)
Hydrogen/electron
NAD+
Niacin(Vit B3)
Hydrogen/electron
Pyridoxal phosphate(PLP)
Pyridoxine(VitB6)
Amino group
Coenzyme A (CoA)
Biotin
Folate coenzymes
Adenosine tri phosphate(ATP)
Pantothenic acid
Biotin
Folic acid
-------
Acyl group
CO2
One carbon group
Phosphate
INORGANIC COFACTOR
(ACTIVATORS)
 Metals/ Inorganic ions
 2 types
Metal activated enzyme
Metallo enzymes
 Metal activated enzymes
 Metal is not tightly bound by the enzyme
 ATPase (Mg2+)
 Enolase(Mg2+)
 Chloride (Cl-) -salivary amylase
 Ca2+ and Pancreatic lipase
INORGANIC COFACTOR
(ACTIVATORS)
 Metallo enzymes
 Metals are tightly bound with enzymes
Alcohol dehydrogenase (zinc)
Carbonic anhydrase (zinc)
DNA Polymerase (zinc)
Xanthine oxidase(molybdenum)
Catalase, peroxidase(Iron)
Cytochrome oxidase(iron)/(copper)
Hexokinase, Pyruvate kinase(Magnesium)
Glutathione peroxidase( selenium)
MECHANISM OF ENZYME ACTION
 Enzymes act by binding substrates & lowering activation energy
(energy needed for reactants to undergo reaction)
 Higher the activation energy, lower the rate
 Transition state- Energy barrier has to be overcome
REACTION COORDINATE
Catalyst for H2O2
decomposition
Energy of activation
(kcal/ mol)
None
18.0
Platinum
11.8
Catalase
4.2
MECHANISM OF ENZYME ACTION
 Lower energy status of transition state is d/t
Substrate strain
Proper orientation of substrates
Proximity of substrates
Change of electrostatic environment around the substrates
Acid – Base catalysis: Proton donors or acceptors
LOWERING OF ACTIVATION ENERGY
 Enzymes lower energy of activation
 Activation energy: Energy needed to convert al molecule of substrates
from ground state to transition state
ACID BASE CATALYSIS
 Specific and general
 Histidine (imidazole group)
 Imidazole pK’ value is 6.0
 Ribonuclease : an example
SUBSTRTAE STRAIN
 Binding of substrate to a preformed site on the enzyme induces strain
in substrate
COVALENT CATALYSIS
 Nucleophilic groups (negatively charge) or electrophilic (positively
charged) group of enzyme attacks substrate
 Nucleophilic groups (negatively charge) at the active site – serine
hydroxyl group, cysteine sulfhydryl group and histidine imidazole group
 Serine class: Trypsin, Chymotrypsin
MICHAELIS- MENTEN THEORY
 Enzyme E combines with a single substrate S to form EnzymeSubstrate complex ES at the active site, which immediately dissociates
to form free enzyme E and the product P
S+ E
ES
E+P
MODELS TO EXPLAIN MECHANISM
OF SPECIFICITY AND CATALYSIS
 Formation of ES complex can be explained by 2 models:
 Fischer’s Lock and Key Model
 Koshland’s Induced Fit Model
MODELS TO EXPLAIN MECHANISM
OF SPECIFICITY AND CATALYSIS
FISCHER’S
LOCK AND KEY MODEL
 Active site of enzyme is pre-shaped & rigid
 Complimentary to the substrate
 Fit exactly into one another like key in lock
 Explains only enzyme specificity
KOSHLAND’S
INDUCED FIT MODEL
KOSHLAND’S
INDUCED FIT MODEL
 Hand in glove Model
 Interaction of S & E induces a conformational change in E like glove
when hand is introduced
 Active site is not rigid
 Binding of substrate induces conformational changes in the enzyme –
leads to precise orientation of the catalytic groups catalysis
 Explains both enzyme specificity and catalysis
NOMENCLATURE OF ENZYMES
 Describe type of reaction & add suffix “-ase”
Dehydrogenases
Proteases
Isomerases
 Modifiers:
Hormone sensitive lipase
Cysteine protease
RNA polymerase III
NOMENCLATURE OF ENZYMES
 International Union of Biochemists (IUB): unique name and 4 digit EC
code number
 Enzyme name has 2 parts:
Names indicating substrate(s) & cofactor
Type of reaction catalyzed ( ends in ‘ase’)
 Additional information in parenthesis
CLASSCATAION OF ENZYMES
 The International Union of Biochemistry and Molecular Biology (IUBMB)
 6 major classes of enzymes
 Mnemonic: OTHLIL
 Oh That Heart Lives Is Lovely
CLASSCATAION OF ENZYMES
 Oxidoreductases
 Transferases
 Hydrolases
 Lyases
 Isomerases
 Ligases






Oxidation-reduction
Transfer of group of atoms
Hydrolysis
Cleavage of bonds without hydrolysis
Rearrangement of atoms
Joining of molecules (using ATP)
OXIDOREDUCTASE
 Catalyzes oxidation of one substrate with simultaneous reduction of
another substrate or coenzyme

Alcohol dehydrogenase
Alcohol
Aldehyde
NAD+
NADH +H+
Malate dehydrogenase

Malate
Oxaloacetate
NAD+
AH2 + B
NADH +H+
A + BH2
TRANSFERASE
 These enzymes catalyze transfer of a group other than H such asamino, phosphoryl, methyl, from one substrate to another
A- X+ B
A + B- X
TRANSFERASE
 Transfer groups from one substrate to another
Hexokinase
Hexose
Hexose-6-phosphate
ATP
ADP
Alanine transaminase
Pyruvate
Glutamate
Alanine
α-ketoglutarate
HYDROLASE
 Catalyze cleavage of a molecule by addition of water (hydrolysis)
 Cleaves ester, peptide, glycosidic bonds
A–B + H2O
A-OH + B–H
HYDROLASE
 Lactase
Lactose + H2O
Glucose + Galactose
 Sucrase
Sucrose + H2O
 Trypsin, Chymotrypsin
Glucose + Fructose
LYASES
 Break bonds by other than hydrolysis
 Split C-C, C-O, C-N, leaving double bonds
Fructose 1, 6 bis phosphate
Aldolase
Dihydroxyacetone phosphate Glyceraldehyde-3-phosphate
 Fumarase
Malate
Fumerate + H2O
ISOMERASE
 Enzymes produce isomers of substrates
 Include racemases, epimerases and cis-trans isomerases.
A
A‘
 Triose phosphate isomerase, Alanine racemase, Retinal isomerase
ISOMERASE
 Phosphohexose isomerase
Glucose 6-P
Fructose P
 Phosphotriose isomerase
Glyceraldehyde 3-P
Dihydroxyacetone 3-P
LIGASE
 These enzymes catalyze synthetic reactions
 Two molecules joined by covalent bond at expenses of high energy
phosphate bond of usually ATP hydrolysis of ATP
 Synthetases
A+B
C
ATP
ADP + Pi
 Glutamine synthetase
Glutamate + NH3 + ATP→ Glutamine + ADP + Pi
LIGASE
 Pyruvate carboxylase
Pyruvate + CO2
Oxaloacetate
ADP + Pi
ATP
Biotin
ATP
Malonyl CoA
Biotin ADP + Pi
 Acetyl CoA carboxylase
Acetyl CoA + CO2
HEXOKINASE
ATP + D – Hexose
ADP + Hexose 6 -P
 E.C.2.7.1.1
 ATP:D-Hexose 6-phosphotransferase
 Class 2: Transferases
 Subclass 7: transfer of a phosphoryl group
 Sub-subclass 1: alcohol is phosphoryl acceptor
 1: the alcohol phosphorylated is hexose
MALIC ENZYME
L-malate + NAD+
NADH + H + + Pyruvate +CO2
 EC.1.1.1.37
 L-malate:NAD+oxidoreductase(decarboxylating)
CREATINE KINASE
Creatine + ATP
Creatine phosphate+ ADP
 EC 2.7.3.2
 ATP: creatine phosphotransferase
 Class 2: Transferases
 Subclass 7: phosphotransferases
 Sub subclass 3: Phosphotransferases with a nitrogenous group as
acceptor
 2: designates creatine kinase
SYNTHASES
 They do not belong to ligases
Glycogen synthase (class II)
ALA synthase (class II)
ATP synthase (class III)
METHODS OF ENZYME ASSAYS
Endpoint method
 readings are taken at the end of reaction
Kinetic method
 readings are taken at different time intervals
ENZYME UNITS
 Rate of reaction catalyzed by the enzyme is proportionate to the
quantity of enzyme present
International unit (I.U)
Katal
Turnover number
Katal
 Number of moles of substrate transformed per second per liter of
sample.
ENZYME UNITS
International unit
 One IU is amount of enzyme that converts 1µmol of substrate per
minute per liter of sample (U/L)
 1 I. U = 16.67 nKat
1 nKat = 0.06 U
Turnover number
 Number of substrate molecules transformed per unit time by a single
enzyme molecule
Specific activity - a measure of purity
 The number of enzyme units present per milligram of protein
ENZYME PURIFICATION
Salting out
Gel filtration chromatography
Ion exchange chromatography
Electrophoresis
INTERNATIONAL UNIT OF
ENZYME ACTIVITY
 Amount that causes transformation of 1µmole of substrate per minute
under optimal conditions
 Excess of substrate
 Optimum pH
 Temperature- @ which enzyme is stable & highly active (25oC)
 Coenzymes and cofactors
SPECIFIC ACTIVITY
 It is the number of enzyme units per mg of protein
 Measure of enzyme purity
 Reaches a maximum value and remains constant
ENZYME SPECIFICITY
 Enzymes are reaction/substrate specific
 More specific than inorganic catalysts
 Specificity is because of d/t
complementary shape
charge characteristics of E & S
 Koshland’s induced fit model explains substrate specificity of enzymes
ENZYME SPECIFICITY
Absolute Specificity
 Certain enzymes act only on one substrate
 Ex: Glucose oxidase>oxidize β D glucose only
Glucokinase
Glucose
Glucose-6-phosphate
 Glucokinase cannot act on galactose
urease
Urea
Ammonia +CO2
ENZYME SPECIFICITY
Bond Specificity
 Act on substrate having specific bonds
 All proteolytic enzymes
 Trypsin & chymotrypsin- hydrolyze peptide bonds formed by carboxyl
groups of Arg, Lys
 Glycosides on glycosidic bond of carbohydrate
 Lipases on ester links of lipids
ENZYME SPECIFICITY
Group Specificity
 Act on substrate having specific groups
 Act on a structurally related substances
 Hexokinase - several hexoses to form respective hexose-6-phosphate
ENZYME SPECIFICITY
Stereo specificity
 Act on only one type of isomers (exception- isomerase)
 Humans enzymes are specific for D–carbohydrates and L-aminoacids
L amino acid oxidase
D amino acid oxidase
ENZYME KINETICS
 Study of all the factors that affect rates of enzyme catalyzed reactions
 The study of rate of enzyme catalyzed reactions & factors affecting
these reaction rates
 study of reaction rate
 determines number of steps involved
 determines mechanism of reaction
 identifies “rate-limiting” step
ENZYME KINETICS
 Enzyme concentration
 Substrate concentration
 Temperature
 pH
 Product concentration
 Presence of coenzymes, activators or inhibitors
KINETIC THEORY
 Only molecules that collide can react
 Energy barrier that must be overcome
 Temperature and reactant concentration
 Higher temperatures – biomolecules are not stable
 Catalysts enhance the local concentration – heterogeneous solution
chemistry
IMPORTANCE OF STUDYING
ENZYME KINETICS
 Understanding mechanism of enzyme action
 Understanding mechanism of enzyme inhibition
 Clinical diagnosis (measuring activity of enzymes helps in diagnosing
disease)
 Understanding disease processes
BASICS
 Catalyst is required in much lower concentration than the substrates
 Similarly in an enzyme catalysed reaction
[E] <<< [S]
 [E] nmoles/liter and [S] mmol/liter
EFFECT OF ENZYME CONCENTRATION
 Initial velocity of enzyme-catalyzed reaction is directly proportional to
enzyme concentration
 As enzyme concentration [E] increases velocity (V) of enzyme reaction
also increases progressively
 straight line is obtained when result is plotted on a graph with V on y
axis and [E] on x axis
GRAPH: EFFECT OF ENZYME
CONCENTRATION ON VELOCITY
y
Velocity (V)
x
Enzyme concentration
EFFECT OF SUBSTRATE
CONCENTRATION
 As substrate concentration [S] is increased, velocity also increases
correspondingly in the initial phases; but the curve flattens afterwards
 If velocity(V) of an enzyme reaction is measured at various [S] & result
is plotted on a graph with V on y axis and [S] on x axis, a rectangular
hyperbolic curve is obtained
GRAPH: EFFECT OF SUBSTRATE
CONCENTRATION ON VELOCITY
y
Vmax------------------------------------------------------------C
Vmax------------- B
Velocity
A
x
Km Substrate concentration
EFFECT OF SUBSTRATE
CONCENTRATION ON VELOCITY
EFFECT OF SUBSTRATE
CONCENTRATION
 A: Substrate concentration is very low –Voα [S]
 B: Substrate concentration is more – Vo is not directly proportional to
[S] – smaller ↑
 C: Substrate concentration is high –Vo is independent of [S] –
vanishingly smaller ↑
 Vmax – Maximal velocity
 Graph approaches, but never reaches a plateau Enzyme is
“saturated” with its substrate
MICHAELIS-MENTEN EQUATION
 Describes the relationship of substrate concentration [S] to velocity
(V) for enzyme catalyzed reactions.
 V = initial velocity
 Vmax = maximum velocity
 [S] = substrate concentration
 Km = Michaelis constant
KM VALUE(Michaelis Constant)
 Vi
 Initial velocity
 The velocity measured when very little substrate has reacted
 Vmax
 Maximal velocity
 The maximum reaction rate attainable in presence of excess substrate
KM VALUE(MICHAELIS CONSTANT)
 The substrate concentration at half the maximal velocity (½ Vmax) in
an enzyme catalyzed reaction
 When V =½ Vmax , Km= [S]
 Denotes that 50% of enzyme molecules are saturated with substrate
molecules at that particular substrate concentration
KM VALUE
 Km value is characteristic feature of a particular enzyme for a specific
substrate
 Constant for an enzyme - signature
 Ranges from 10-5 to 10-2 moles/liter
KM VALUE: SIGNIFICANCE
 A measure of affinity of enzyme for substrate
-low Km value indicates a strong affinity
-high Km reflects a weak affinity
 Helps to know natural substrate of an enzyme having more than one
substrates
 Helps to study of mechanism of enzyme inhibition
 Km values of isoenzymes are different for the same substrate
HEXOKINASE & GLUCOKINASE
 Hexokinase is present in all cells except hepatocytes and β-cells of
pancreas
 Glucokinase is present in hepatocytes and β-cells
 Hexokinase has low Km for glucose (0.05mmol/L) – ensures supply
even at low blood glucose
 Glucokinase – high Km value (10 mmol /L) – removes glucose after a
meal from portal vein
HEXOKINASE & GLUCOKINASE
ALGEBRAIC TRANSFORMATION
 Double reciprocal plot
 Vmax is only approached and never attained
 Determination of Km value at a [S] lower than Vmax
 Algebraic transformation of Michaelis – Menton equation
Vi = Vmax[S]
Km + [S]
LINEWEAVER BURK EQUATION
LINEWEAVER BURK EQUATION
Equation for a straight line
y = ax + b
y= 1/Vi
x = 1/[S]
a = Km/Vmax (Slope)
b = 1/Vmax (y intercept)
ALGEBRAIC TRANSFORMATION
 Setting y = 0,
 0 = ax + b
 -b = ax
 x = -b/a
 x = - 1/Vmax . Vmax/Km = -1/Km
 Km = a / b = Km/Vmax ÷ 1/Vmax = Km
 Lineweaver – Burk equation is a mathematical expression of the shape
of the rectangular hyperbolic curve
 Plot 1/[S] vs 1/Vo (L-B equation for straight line)
ORDER OF RECTION
 Rate constant (k) measures how rapidly a reaction occurs
k1
A
B+C
k-1
 Rate (v, velocity) = (rate constant) (concentration of reactants)
v= k1 [A]
 1st order rxn (rate dependent on concentration of 1 reactant)
v= k-1[B][C]
 2nd order rxn (rate dependent on concentration of 2 reactants)
 Zero order rxn (rate is independent of reactant concentration)
EFFECT OF TEMPERATURE
ON ENZYME ACTIVITY
 Velocity (V) of an enzyme-catalyzed reaction increases when
temperature of the medium is increased, reaches a maximum and then
falls
 A graph with V on y axis and temperature on x axis, a bell-shaped curve
is obtained
 ↑Temp: -increases kinetic energy of molecules
Increases collision frequency
Lowers energy barrier
EFFECT OF TEMPERATURE
ON ENZYME ACTIVITY
Optimum temperature
Velocity
Low
37oC
Temperature
High
EFFECT OF TEMPERATURE
ON ENZYME ACTIVITY
 Temperature at which the velocity is maximum is - Optimum
temperature (37oc for most enzymes in humans)
 If temperature is very high (>55oC)- heat denaturation activity of the
enzyme decreases
Exception: Thermus acquaticus enzymes are stable and active even in
1000C Used in PCR
Q10 (temperature coefficient)
 The factor by which the rate of reaction increases for every 100 C rise
in temperature.
 Q10 = 2
EFFECT OF pH
ON ENZYME ACTIVITY
 Velocity V of an enzyme-catalyzed reaction is measured at various pH
values and plotted ,a bell-shaped curve is obtained
 Enzyme have an optimum pH on both sides of which the velocity will be
drastically reduced
 pH changes will cause alteration in charged state of enzyme/
substrate or both
EFFECT OF pH
ON ENZYME ACTIVITY
Optimum pH
Velocity
pH
EFFECT OF pH
ON ENZYME ACTIVITY
 The pH at which the velocity is maximum is called the optimum pH
 Usually 6 – 8 for most human enzymes
 Exceptions:
Pepsin (1-2)
Alkaline phosphatase (9-10)
Acid phosphatase (4 - 5)
 At extreme pH, enzyme gets denatured - activity is drastically reduced
PRODUCT CONCENTRATION
 An increase in [P] decreases velocity of the enzyme-catalyzed reaction
ENZYMES WITH ≥2 SUBSTRATES
 Different Km value for each substrate
 Single displacement reaction
Creatine kinase
Malate dehydrogenase
 Double displacement reaction (Ping – pong reaction)
AST
ALT
ENZYMES WITH ≥2 SUBSTRATES
E + A + B <-> E + P + Q
 Sequential Reactions
ordered
random
 Ping-Pong Reactions
 Cleland Notation
SEQUENTIAL REACTIONS
 Ordered
A
E
 Random
EA
A
Q
P
B
(EAB)
(EPQ)
P
B
EA
EQ
E
Q
EQ
(EAB)(EPQ)
E
E
EB
B
EP
A
Q
P
PING PONG REACTIONS
A
E
(EA)(FP)
P
B
(F)
Q
(FB)(EQ)
 First product released before second substrate binds
 When E binds A, E changes to F
 When F binds B, F changes back to E
E
ENZYME INHIBITION
 Enzyme inhibitor:
 A substance which binds to the enzyme and decrease the velocity of
the enzyme-catalyzed reaction
 Enzyme inhibition :
 Reduction of enzyme activity by binding of inhibitor to the enzyme
ENZYME INHIBITION
 Inhibitor reduces or abolishes enzyme activity by combining with the E
or ES complex
 Substrate specific
 Nature of functional groups at active site
 Function groups involved in active conformation
 Regulation of intermediary metabolism
 Drugs used in medicine
INHIBITION:SIGNIFICANCE
 Used to elucidate the mechanism of enzyme action
 Many drugs and poisons are enzyme inhibitors
Anticancer
Antibiotics
Antivirals
Immunosuppresents
ENZYME INHIBITION
 Organic and inorganic inhibitors
Irreversible inhibitors
Reversible inhibitors
 3 major types of reversible inhibition
Competitive
Noncompetitive
Uncompetitive
 Distinguished by reaction kinetics – only for reversible reactions
ENZYME INHIBITION
 Inhibitor may bind to
Active site or
Site other than the active site
may be to a Free enzyme molecule or ES-complex
 Inhibitor binds to specific R-groups of amino acid residues, involved in
 Catalysis
 Substrate-binding
 Maintenance of functional conformation of the enzyme
ENZYME INHIBITION
INTERACTIONS BETWEEN ENZYME
AND INHIBITOR
 Weak, non-covalent bonds - Reversible inhibition
Ionic bond
H-bond
Hydrophobic bond
 Strong covalent bond: irreversible inhibition
 Enzyme – Inhibitor complex is formed (EI complex)
ENZYME INHIBITION
2 Types
Basis : Stability of EI complex
Reversible
Irreversible
 Weak non-covalent bonds
 Easily dissociable
 Enzyme activity is restored by removing inhibitor
Competitive
Non competitive
Uncompetitive
REVERSIBLE INHIBITION
 COMPETITIVE INHIBITION:
 Combines with the free enzyme at active site
 Resembles substrate – substrate analogue
 E + I ↔ EI
 Inhibitor constant, ki = [E][I] / [EI] = dissociation constant
 ki value is inversely related to the efficiency of inhibition
 Type of reversible inhibition
 Structural analog Competes with the substrate for active site
COMPETITIVE INHIBITION
 At sufficiently high [S], the [EI] is vanishingly small  ‘S’ displaces ‘I’
from substrate binding site  overcome the inhibition
 Inhibition can be relieved by increasing substrate concentration
 At infinitely high [S]
(1/[S] = 0)
Vi is the same as in the absence of inhibitor
Competitive inhibitor does not change Vmax
 Does not interfere with breakdown of ES complex to form product
 1/K’m is smaller than 1/Km
 Competitive inhibitor increases apparent Km (K’m)
a) Competitive inhibition
Substrate
Active site
Enzyme -Substrate
Complex
Enzyme
Catalysis
Structural analog
Competitive
Inhibitor
Enzyme -product
Complex
No catalysis
Products
Enzyme - Inhibitor complex
Enzyme
Active site
Enzyme
Substrate
S
Substrate
S
S
S
Increase in [S]
Inhibition is reversed
Enzyme is active
Competitive
Inhibitor
Enzyme -product
Complex
Catalysis
Enzyme - Inhibitor complex
Products
No catalysis
EScomplex
EI
complex
1. Competitive inhibition
Enzyme
COMPETITIVE INHIBITION
 Michaelis Menten plot: (Substrate-saturation curve)
Vmax
With inhibitor
Km
Km
 Vmax remains unchanged
 Km increases.
COMPETITIVE INHIBITION
REVERSIBLE INHIBITION
 X intercept = -1/Km. α
 Slope = Km. α/ Vmax
 Inhibition of succinate dehydrogenase by Malonate
 Malonate: -OOC – CH2 – COO-
COMPETITIVE INHIBITION
 Sulfonamide: Structurally similar to PABA Inhibits synthesis of folic
acid in bacteria Antibiotic
 Folic acid: Pteridine – PABA – Glutamic acid
 Ethanol in the treatment of methanol poisoning
 Methanol is component of antifreeze – causes blindness
 Methanol → formaldehyde (damages eyes)
 Ethanol → acetaldehyde compete for alcohol dehydrogenase
COMPETITIVE INHIBITION
 Methotrexate: Dihyrdofolate reductase natural substrate is folic
acid anticancer drug
 Dicumarol: Inhibits Vit K 2,3-epoxide reductase  Inhibits regeneration
of active form of Vitamin K
 Isonicotinic acid hydrazide (INH, Isooniazid)  Inhibits Pyridoxal
kinase - Antituberculosis drug
 Physostigmine: Acetylcholine esterase Rx Myesthenia gravis
 α-Methyl DOPA: DOPA decarboxylase antihypertensive drug
decreases the production of epinephrine
 Loastatin: HMG CoA Reductase  Hypocholesterolemic drug
NON COMPETITIVE INHIBITION
 Inhibitor has no structural resemblance with S
 Binds at a site other than active site (EI, ESI)
 Does not compete with substrate for active site
 Slows the decomposition of ESI complex to product
 Alters conformation of enzyme  effects catalysis but not substrate
binding
 Decreases Vmax
 Km remains unaffected
 At high [S], ‘S’ cannot displace the ‘I’ molecule from substrate binding
site and thus cannot overcome the inhibition
NON COMPETITIVE INHIBITION
 Enzymes which require metal ions inhibited by EDTA
 Trypsin inhibitors from soybean and ascaris
 Carbonic Anhydrase by Acetazolamide (diuretic, glaucoma)
MICHAELIS-MENTEN PLOT
(SUBSTRATE-SATURATION CURVE)
Vmax
With inhibitor
KmK
m
 Decreases Vmax
 Km remains unaffected
Process of Non competitive inhibition
Substrate
ES Complex
EP Complex
Catalysis
Active site
Enzyme
Inhibitor
No Catalysis
Substrate
EI Complex
ESI Complex
UNCOMPETITIVE INHIBITION
 Reversible inhibition
 Inhibitor combines with ES complex to form inactive ESI complex
 Prevents ES from proceeding to E + P or back to E + S
 ES + I ↔ ESI
 Vmax decreases
 Apparent Km decreases
 Increased [S], cannot overcome the inhibition
 Inhibition of alkaline phosphatase by phenylalanine
 Occurs with multi substrate enzymes
UNCOMPETITIVE INHIBITION
 Slope remains constant (Km/ Vmax)
 X intercept: - α / Km, Y intercept: α / Vmax
 Vmax decreases
 Apparent Km decreases
MICHELIS-MENTEN PLOT :
(SUBSTRATE-SATURATION CURVE).
Vmax
Vmax. i
 Vmax decreases
 Apparent Km decreases
Without inhibitor
With inhibitor
Process of Uncompetitive inhibition
Substrate
ES Complex
EP Complex
Catalysis
Active site
Enzyme
Inhibitor
No Catalysis
ESI Complex
REVERSIBLE INHIBITION
Inhibitor
Binding site
Increase [S]
Binding to ES
Vmax
Km
Structural analog Not a structural
of Substrate
analog
Active site
Active/other site
Reverse inhibition Cannot reverse
Not possible
possible
Kinetics in presence of inhibitor
Unchanged
decreased
Increased
same
Not a structural
analog
Any other site
Cannot reverse
Binds only to ES
decreased
decreased
IRRIVERSIBLE INHIBITION
 Binds to the enzyme tightly by covalent bonds
 Forms stable complex (EI complex)does not dissociate significantly
 Permanently modifies a functional group required for catalysis, binding
or functional conformation
 Enzyme poisons
 Cannot be studied by Michaelis – Menten principles
 Poisons (toxic substances)
 Oxidizing agents
IRRIVERSIBLE INHIBITION
 Affinity labels:
 Substrate analogs that possess a highly reactive group that is not
present on the natural substrate
 DIFP reacts with Ser195 of chymotrypsin
 DIFP also inhibits acetyl cholinesterase, trypsin
IRRIVERSIBLE INHIBITION
 Mechanism based (suicide inhibitors):
 Substrate analogs transformed by catalytic action
 Inactive initially  Binds to the active site of the enzyme  modified
effective inhibitor  Product combines covalently with active site
inhibits further reactions of same enzyme
 Allopurinol – xanthine oxidase – Alloxanthine
 Di fluro methyl ornithine (DFMO) - Ornithine decarboxylase-Treatment
of Trypanosomiasis (sleeping sickness)
 Ornithine → Putrescine → → → Polyamine
IRRIVERSIBLE INHIBITION
 Transition state analogs:
 Resembles transition state of natural substrate
 Do not covalently modify the enzyme
 Penicillin – Trans peptidase
IRRIVERSIBLE INHIBITORS
THERAPUTIC APPLICATION
Inhibitor
Aspirin
Allopurinol
5-Fluorouracil
Penicillin
Pargyline
Target enzyme
Cyclooxygenase
Xanthine oxidase
Thymidylate synthetase
Transpeptidase
Monoamine oxidase
Effect / Application
Anti-inflammatory
Gout
Anticancer
Antibacterial
Antihypertensive
IRRIVERSIBLE INHIBITORS
APPLICATIONs
INHIBITOR
Fluoride
ENZYME
Enolase
Cyanide
Cyt. Oxidase
OP compounds ACh esterase
Malathion
Heavy metal ion
Hg, Pb, Arsenic
GROUPS ON THE
IMPORTANCE /
ENZYME
APPLICATION
Removes Mg2+ from Inhibits glycolysis
active site
Respiratory poison
Serine group in the Insecticides
active site
Covalent bond with Poison
-SH groups
Substrate
Active site
Enzyme
ES Complex
Substrate
analog
Inactive
inhibitor
Enzyme
- Substrate analog
complex
Mechanism based inhibition
No catalysis
Catalysis
Enzyme substrate analog complex
Enzyme –
Inhibitor
complex
Active
inhibitor
Enzyme – Inhibitor complex
IRRIVERSIBLE INHIBITORS
THERAPUTIC APPLICATION
Allopurinol
Hypoxanthine
Xanthine
Xanthine Oxidase
Uric acid
Xanthine Oxidase
Structural analog ↑↑↑Uric acid leads to GOUT
Alloxanthine
Allopurinol
Xanthine Oxidase
Clinical use of Allopurinol
↓↓ uric acid production Treatment of gout
IRRIVERSIBLE INHIBITORS
THERAPUTIC APPLICATION
 5 - Flurouracil
 Inhibits Thymidine kinase
 Synthesis of Thymidine triphosphate for synthesis of DNA
 Anticancer drug
 Fluroacetate
 Aconitase(of TCA cycle)
REGULATION OF ENZYMES
 Homeostasis – Constant intracellular environment
 Change in substrate concentration
 Unidirectional flow of metabolites
 Compartmentation ensures metabolic efficiency
 “bottleneck” or “rate-limiting reaction” or “flux generating steps of
metabolic pathways”
 Regulation by changing
Quantity
Catalytic efficiency
REGULATION OF ENZYMES
 Homeostasis – Constant intracellular environment
 Change in substrate concentration
 Unidirectional flow of metabolites
 Compartmentation ensures metabolic efficiency
 “bottleneck” or “rate-limiting reaction” or “flux generating steps of
metabolic pathways”
 Regulation by changing
Quantity
Catalytic efficiency
REGULATION OF ENZYMES
 Enzyme quantity – regulation of gene expression (Response time =
minutes to hours)
Transcription
Translation
Enzyme turnover
 Enzyme activity (rapid response time = fraction of seconds)
Allosteric regulation
Covalent modification
Association-disassociation
Proteolytic cleavage of proenzyme
REGULATORY ENZYMES/
KEY ENZYMES
 Catalyze committed step in metabolic pathway
 Reactions are irreversible
 These reactions are rate-limiting for whole pathway.
 Regulatory enzymes are either at or near the initial steps in a pathway
Or
 Part of a branch point or cross-over point between pathways
MECHANISMS FOR REGULATION OF
ENZYME ACTIVITIES
(i) Change of enzyme
concentration
(ii) Change of enzyme
catalytic efficiency
Change in rate of enzyme
Synthesis
Change in rate of enzyme
degradation
↑↑↑ Synthesis
Enzyme Induction
↓↓↓ Synthesis
Enzyme Repression
MECHANISMS FOR REGULATION OF
ENZYME ACTIVITIES
(ii) Change of enzyme catalytic efficiency
Non-covalent Modification
(Reversible, Allosteric Enzymes)
Reversible
PhosphorylationDephosphorylation
Covalent Modification
Irreversible
Limited Proteolysis
CONTROL OF
ENZYME CONCENTRATION
CONTROL OF ENZYME SYNTHESIS:

DNA
↑↑↑
↓↓↓
Transcription
↑↑↑
mRNA ↓↓↓
Translation
Protein (Enzyme)
Inducers
Hormones, other molecules
 Repressors
Enzyme
Enzyme Induction
Repression
↓↓↓
↑↑↑
INDUCTION/ REPRESSION
 Induction : ↑↑↑ synthesis of enzyme through ↑↑↑ gene transcription
of the relevant mRNA, leading to an ↑↑↑ in [E]
 Inducible enzymes / Adaptive enzymes
 Repression : ↓↓↓ synthesis of enzyme by ↓↓↓ gene transcription of
the relevant mRNA, leading to a ↓↓↓ in the [E]
INDUCTION/ REPRESSION
Enzyme
Glucokinase
ALA synthase
(Heme synthesis)
HMG CoA reductase
(Cholesterol synthesis)
Transaminases- AST, ALT
Inducers
Repressor
Insulin
-
Glucagon
Heme
-
Cholesterol
Glucocorticoids
-
CONTROL OF
ENZYME DEGRADATION
 Lysosomes / proteosomes
 Ubiquitin proteosome pathway
 26S proteosome – 30 polypeptides – hollow cylinder
 E3 ligases
 Regulation: involves transfer of a polypeptide, ubiquitin, to targeted
enzymes (proteins).
ALLOSTERIC REGULATION
 Allosteric enzymes: activity can be altered by binding of an effector/
modifier at a site other than the catalytic site called allosteric site
(allo=other, stereos=space)
 Allosteric enzyme has
Catalytic site - substrate binding and catalysis
Allosteric site – modifier binding
 Brings about conformational change in active site
 # first committed step (irreversible) of a biosynthetic sequence
 V-series: catalytic efficiency is altered (↓Vmax)
 K-series: affinity of the enzyme for the substrate is altered(↑ Km)
ALLOSTERIC ENZYMES AND ALLOSTERIC REGULATION
Binds to
Active site
Catalysis
Substrate
Allosteric site
Binds to Modifier / Effector
↑↑ activity
Positive Modifier (+)
Allosteric activator(+)
↓↓ activity
Negative Modifier (-)
Allosteric inhibitor(-)
ALLOSTERIC REGULATION
Allosteric enzyme
Active site
Substrate
S
ES complex
Enzyme
EP complex
Allosteric site is altered
Active site is altered
Allosteric site
E
↑↑
Effector
E
Binding either S or E conformational change in the
enzyme
ALLOSTERIC REGULATION
Allosteric enzyme
Substrate /
End Product
starting material
S
A
B
C
D
E
F
P
E1
E2
E3
E4
E5
E6
E7
↑↑


↑↑
•starting material / an early
intermediate
•feed forward activation
•end product
•feedback inhibition
ALLOSTERIC REGULATION: EX
Allosteric Allosteric
Allosteric Enzyme
Pathway
activator inhibitor
Phospho fructokinase
Glycolysis
AMP
ATP, Citrate
ALA synthase
Heme synthesis
Heme
HMG CoA reductase
Cholesterol synthesis
Cholesterol
Acetyl CoA carboxylase Fatty acid synthesis Citrate
Acyl CoA
PHOSPHOFRUCTOKINASE( PFK)
Fructose-6-P + ATP -----> Fructose-1,6-bisphosphate + ADP
 PFK catalyzes 1st committed step in glycolysis (10 steps total)
 (Glucose + 2ADP + 2 NAD+ + 2Pi  2pyruvate + 2ATP + 2NADH)
 Phosphoenolpyruvate is an allosteric inhibitor of PFK
 ADP is an allosteric activator of PFK
ALLOSTERIC ENZYMES AND
ALLOSTERIC REGULATION
 Allosteric enzymes usually have 4o structureOligomeric proteins –
more than one S binding site
 Vo vs [S] plots (Substrate saturation curve) sigmoidal for at least
one substrate
 Bacterial aspartate transcarbamoylase is inhibited by CTP, but
activated by ATP
 First reaction of pyrimidine biosynthesis
 Corresponding mammalian enzyme is not allosteric
 Carbamoyl-P + L-Aspartate → Carbamoyl Aspartate
ALLOSTERIC ENZYMES AND
REGULATION
 Vo vs [S] plots give sigmoidal curve for at least one substrate
 Binding of allosteric inhibitor or activator does not effect Vmax, but
does alter Km
 Allosteric enzyme do not follow M-M kinetics
REGULATION BY
COVALENT MODIFICATION
 Reversible covalent modification
 Irreversible covalent modification
Reversible covalent modification:
Enzyme
Active form
Inter-convertible
Enzyme
Inactive form
Addition/ Removal of groups
Phosphorylation / Dephosphorylation
( Adenylation / deadenylation)
PHOSPHORYLATION
DEPHOSPHORYLATION
 Short-term, Readily reversible
 Some enzymes are active either in phosphorylated form OR in dephosphorylated form
ATP
Protein Kinase
ADP
Enzyme
Enzyme - P
De-phosphorylated
enzyme
Pi Phosphatase H2O
Phosphorylated
enzyme
Ser, Thr, Tyr
COVALENT MODIFICATIONS
Enzyme
Glycogen Phosphorylase
Glycogen synthase
Acetyl CoA carboxylase
Hormone sensitive lipase
Pyruvate dehydrogenase
Active form Inactive form
EP
E
E
EP
E
EP
EP
E
E
EP
 Methylation/ Adenylation, , Phosphorylation
 Protein kinases, protein phosphatases
 Seryl, threonyl or tyrosyl residues
IRREVERSIBLE
COVALENT MODIFICATION
 Activation of Zymogens or Proenzymes
Zymogens /
Proenzymes:
Inactive precursors of
enzymes
Partial proteolysis
Enzymes
Active enzymes
Removal of Peptide
Catalytic site is exposed
 This type of modification is irreversible
IRREVERSIBLE
COVALENT MODIFICATION: EX
 In stomach
Pepsinogen
HCl

Autocatalysis
 In Pancreas
Trypsinogen
 In blood
Prothrombin
Enterokinase

Autocatalysis
Ca++
Thrombin
Pepsin
Trypsin
Clotting of blood
PROTEOLYTIC CLEAVAGE OF
PROENZYME(ZYMOGEN)
PROTEOLYTIC CLEAVAGE OF
PROENZYME(ZYMOGEN)
 Clotting involves series
of zymogen activations
 Seven clotting factors
are serine proteases
involved in clotting
cascade reaction
IRREVERSIBLE
COVALENT MODIFICATION
 Significance
Rapidly ↑↑ [E] in response to physiological demand
Prevents auto digestion/ self-destruction of tissues
Pancreatitis
Regulation is slower than allosteric regulation
 Phosphorylation /dephosphorylation - MC covalent modification
 involve protein kinases/phosphatase
 PDK inactivated by phosphorylation
 Amino acids with –OH groups are targets for phosphorylation
ENZYME COMPARTMENTALIZATION
 Mitochondria: Enzymes of - TCA cycle
Beta oxidation of fatty acids
Electron transport chain
 Cytosol: Enzymes ofGlycolysis
Glycogenesis
Glycogenolysis
Fatty acid synthesis
 Advantages:
 ↑↑[S] in location where the reaction occurs enhances efficiency
 It allows better regulation of the pathways
ENZYME ASSOCIATION/
DISASSOCIATION
 Acetyl-CoA Carboxylase
Acetyl-CoA + CO2 + ATP  malonyl-CoA + ADP + Pi
 First committed step in fatty acid biosynthesis
 In presence of citrate activated
 In presence of fatty acyl-CoA inactivated
citrate
polymerized
unpolymerized
Fatty acyl-CoA
MULTI-ENZYME COMPLEX
 Enzymes associated with metabolic pathway  organized into
macromolecular complexes
 Active only in complex form, and not individually
Pyruvate dehydrogenase
α-Ketoglutarate Dehydrogenase
MULTI-FUNCTIONAL ENZYMES
 Single polypeptide chain  Arranged into many catalytic sites 
each having a different enzyme activity
 Fatty acid synthase complex
 Increased efficiency of the pathway by ↑local [S]
ISOZYMES
 Physically different forms of the same catalytic activity
 Differ in net charge, resistance to denaturing agents, heat stability,
susceptibility to inhibitors & in kinetic constants(Km, Vmax inhibition)
 May be present in different
(a) organisms
(b) tissues of the same organism
(c) cell types
(d) subcellular compartments
(e) within a prokaryote
 Useful in diagnosis, Separated by electrophoresis
ISOZYMES
 Oligomeric proteins
 Usually 2 types of polypeptide chains (subunits) in different
combination isoenzymes differ in their
electrophoretic mobility
optimum pH
heat stability
cofactor requirements
immunological (antibody) response
EXAMPLES OF ISOZYMES
 Lactate dehydrogenase
(LDH) – 5 isoenzymes
 Creatine phospho kinase (CPK / CK)– 3 isoenzymes
 Alkaline phosphatase
(ALP)– 6 isoenzymes
 Aspartate aminotransferase (AST) – 2 Isoenzymes
(Cytoplasmic
Mitochondrial)
LACTATE DEHYDROGENASE
 Wide distributionCells of cardiac, skeletal muscle, liver, kidney,
brain, erythrocytes
 Tetrameric protein
 Two types of subunits –
M (muscle type)
H (heart type)
CH3
I
H - C - OH Lactate
I
+
NAD
COOH
CH3
I
LDH
Pyruvate C = O
I
+
NADH + H
COOH
ISOZYMES
LDH-1
Subunit
makeup
HHHH
LDH-2
Iso enzymes
Tissue of origin Diagnostic Enzymology
Heart
↑↑ Myocardial infarction
HHHM
RBC
-
LDH-3
HHMM
Brain
-
LDH-4
HMMM
Liver
-
LDH-5
MMMM
Liver
↑↑ Muscular dystrophy
Skeletal Muscle
LDH ISOZYMES IN SERUM
 LDH 1 – 25% (Heart muscle)
 LDH 2 – 35% (RBC)
 LDH 3 – 27%
 LDH 4 – 8%
 LDH 5 – 5%
 LDH 1 is inhibited powerfully and LDH 5 weakly by pyruvate
 Flipped pattern in MI
 Separated by electrophoreis
ISOZYMES : GENESIS
 True isozymes: products of different genes, differ in primary structure
Mitochondrial and cytosolic malate dehydrogenase
Glucokinase and hexokinase
 Hybrid isozymes: different subunits in various combinations
LDH, CK
 Allozymes or allelozymes: Same locus of gene have different alleles
Glucose 6P dehydrogenase
 Polymorphism: more than 1% of polymorphism
 Isoforms: Post-translational modification
Alkaline phosphatase
CREATINE PHOSPHOKINASE (CPK)
CREATINE KINASE (CK)
 Cardiac, skeletal muscle, brain
 Dimeric protein
 Two types of subunits –
M (muscle)
B (Brain)
 Isoenzymes : 3
Phospho creatine
ADP
CPK
Creatine
ATP
ISOZYMES
Isoenzymes Subunits
Tissue
Diagnostic Use
CPK1
BB
Brain
CPK2
MB
Heart
↑↑ Myocardial Infarction
CPK3
MM
Skeletal muscle
↑↑ Muscular dystrophy
SIGNIFICANCE OF ISOENZYMES
 Estimation of the serum/plasma levels (activities) of isoenzymes
Diseased tissue (especially necrosis) can be identified
Diagnosis and prognosis of several diseases
 LDH-1
 CPK-2
 CPK-3



Myocardial infarction
Myocardial infarction
Muscular dystrophy
CLINICAL APPLICATIONS:
ENZYMES
 Diagnostic, Therapeutic, Laboratory
 Diagnostic applications
 Functional enzymes (plasma derived enzymes): actively secreted into
plasma, substrates are present in plasma, activity higher in plasma
than cells
Enzymes of blood coagulation
 Non-functional enzymes: cellular enzymes
Physiological – wear and tear
Pathological – in necrosis or increased production
SI UNIT OF ENZYME ACTIVITY
 International unit: One IU is amount of enzyme that converts 1µmol of
substrate per minute per liter of sample (U/L)
 Katal: The amount that catalyses the transformation of one mole of the
substrate per second (kat or k)
 Turnover number: number of substrate molecules transformed per
unit time by a single enzyme molecule
ISOZYMES
Functional enzymes Non functional enzymes
Function in plasma
Specific function
No specific function
Concentration in plasma
Large
Small
Clinically important
decreased
Increased
when plasma levels are
necrosis/ destruction of
Clotting factors
Examples
tissues from which
Ceruloplasmin
enzyme originates
PLASMA ENZYME ACTIVITY
Low plasma
enzyme levels
Blood
vessel
↑ ↑ ↑ ↑ plasma
enzyme levels
Normal wear and
Wear & tear of cancer
Tissue
tear of tissue cells
cells
cells
 Higher than normal
 Less than normal
↑ cell damage
↓ Synthesis
↑ cell Proliferation
Inherited deficiency
DIAGNOSTIC ENZYMES
Diagnostic enzyme
Major diagnostic use
Normal Range
Enzymes which increase in disease conditions
ALT / (SGPT)
Liver diseases
0- 45 IU/L
AST / (SGOT)
MI, Liver disease
0- 40 IU/L
CPK / (CK)
MI, Muscle disorders, crush injuries
10-50 IU/L
LDH
MI, Muscular dystrophy
100-225 IU/L
ALP
Obstructive jaundice, Bone/ Liver ds
30-85 IU/L
Acid Phosphatase
Prostate cancer
1- 5 units/L
Amylase
Acute pancreatitis, Mumps
< 85 IU/L
Lipase
Acute pancreatitis
< 150 units/L
GGT
Chronic alcoholism detection, Liver ds < 30 units/L
DIAGNOSTIC ENZYMES
Enzymes which decrease in disease conditions
Ceruloplasmin (serum ferroxidase)
Wilson’s disease
25-43
(decreased)
mg/dL
 ENZYME PROFILE IN DISEASES
 For accurate diagnosis of a particular disease more than one serum
enzyme are routinely estimated, instead of a single enzyme
ENZYME PROFILE IN DISEASES
Disease
Myocardial
Infarction
Liver disease
Enzymes
1) CPK-2 (CK-MB)
2) AST
3) LDH-1 (H4)
1) ALT
2) AST
3) ALP- increase is seen in obstructive jaundice /
infective hepatitis / alcoholic hepatitis / HCC
4) GGT- is useful in diagnosis of alcoholic liver disease
5) LDH-5 (M4)
ENZYME PROFILE IN DISEASES
Disease
Enzymes
Muscle disorders 1) CK-3 (MM)
2) LDH-5 (M4)
3) AST
4) Aldolase
Bone diseases
1) Alkaline Phosphatase
• Paget’s disease
• Rickets
• Osteomalacia
• Osteoblastoma
MI: CARDIAC MARKERS:
CK-MB
AST
LDH
Myoglobin
Cardiac specific troponins T and I
CK-MB
 Detectable 4-6 hrs post-injury
 Peak @ 12hrs
 Basal levels at 48 – 72hrs
 Serial estimations
 RI = CK-MB mass / Total CK activity X 100 (RI) ≥ 2.5 is suggestive
 Not found in RBCs – not affected by hemolysis
 Mean percentage in blood: MM (CK3) - 80% ,
(CK1) – 1%
MB (CK2) – 5%,
BB
AST
 Rises in 12hrs
 Peaks at 24hrs
 Returns to normal in 3 to 5 days
 Not a reliable cardiac marker – rarely used in diagnosis
 It can reflect diseases of lung, liver or skeletal muscle
 2 isozymes – cytoplasmic and mitochondrial
LDH
 Normal conditions: serum LDH2 is higher than LDH1
 AMI: “LDH flip” – LDH1 higher than LDH2
 Rises by 12-18hrs
 Peaks at 48-72hrs
 Rremains elevated up to 10 days post-infarction
 Not useful in early diagnosis
 Useful in diagnosis after 24hrs of infarction
 Nonspecific: hemolysis, megaloblastic anemia, liver, renal and skeletal
muscle diseases
MYOGLOBIN
 Marker with lowest molecular weight
 Detected after 1-4hrs
 Peaks at 4–12hrs
 Cleared within 24hrs
 Useful early marker
 Nonspecific – present in skeletal and smooth muscles
TROPONINS
 Most specific and sensitive commercially available cardiac marker
 Exists in 3 forms: C, I, T
 In MI, > 20 times higher than the upper reference limit
 Rise 3 – 6hrs (same as CK-MB)
 Remain elevated for 7-10 days after MI
 Troponin I more sensitive than T within 6 hours
 Cardiac markers are negative 12 hours after symptom onset Not MI
AST/ALT
 Not specific to the liver
 AST: liver, heart, skeletal muscle, pancreas, kidney, RBC
 ALT: liver and kidney
 ALT: more specific to liver disease (Hepatocellular damage)
 AST:ALT
 >1: Alcoholic liver disease, nonalcoholic cirrhosis, Reye’s syndrome
 <1: Acute liver diseases
 >2:1 is suggestive while a ratio of 3:1 is highly suggestive of alcoholic
liver disease
ALP
 pH optimum between 9 and 10
 Zn is a constituent ion
 Present in liver, biliary tree, bone, placenta, intestine, kidney, WBC
 Clinical elevations – hepatobiliary or bone
 Hepatic: Intrahepatic cholestasis, Alcoholic hepatitis, Extra hepatic
cholestasis(10– 12 X), Parenchymal liver diseases (2 -3 X)
 Bone disease: (10 – 25 X)Fractures, Paget’s disease, Rickets,
Osteomalacia, Osteoblastic bone tumors
ALP ISOZYMES
 Alpha-1: epithelial cells of biliary canaliculi, increased in obstructive
jaundice
 Alpha-2: heat labile at 65oC for 30 min, stable at 56oC  hepatic cell
– ↑ in hepatitis
 Alpha-2 heat stable at 65oC, inhibited by phenylalanine, placental origin
 Regan enzyme (Carcinoplacental isozyme): similar to placental form,
carcinoma of lung, liver gut, and smokers
5’ NUCLEOTIDASE
 Nucleotide phosphatase
 Nucleotides → Nucleosides
 Marker enzyme for plasma membranes
 High elevation in biliary obstruction
 Moderate elevation in hepatitis
THERAPEUTIC APPLICATIONS OF ENZYMES
OR THERAPEUTIC ENZYMES:
 Enzymes can be administered to patients for treatment purpose.
 Streptokinase/ urokinase/ tissue plasminogen activator(tPA) protease Dissolve clots in MI & Deep Vein Thrombsis (DVT)
Streptokinase / tPA / Urokinase
Plasminogen
Fibrin
(of blood Clot)
Plasmin
low molecular weight
soluble products
 Asparaginase - used in the treatment of leukemia
 Trypsin and lipase –in treatment of Pancreatic insufficiency
ENZYMES IN LABORATORY
 Estimation of concentrations of analytes in body fluids
(plasma/CSF)
 Principle
Specific enzyme
Reagents
Substances
 Measured by colorimetry
Substances estimated
Plasma glucose
Serum cholesterol
Serum triglycerides
Blood Urea
Product
Colored compound
Enzymes used
Glucose oxidase and peroxidase
Cholesterol oxidase
Lipase
Urease
ENZYMES IN LABORATORY
 Estimation of concentrations of certain substances in body fluids
(plasma, CSF etc)
 DNA analysis techniques
 Restriction endonucleases, DNA polymerases , DNA ligases
 Use : diagnosis of genetic diseases, infective diseases, etc.
 ELISA technique
 Utilizes enzymes along with antibodies in measurement of certain
proteins - hormones, viruses
THANK U