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ENZYMES
Dr Nithin Kumar U
Assistant professor
Biochemistry
Yenepoya medical college
Enzymes: Basics
Rate of a Reaction
Reversible & Irreversible Reactions
Reaction Equilibrium
Catalysis
Background: A reaction
r1
A+B
C+D
Reaction rate:
r1 α [A] [B]
Therefore,
r1 = k1 [A] [B]
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 = r 2
Background
r1 = k1 [A] [B]
&
r2 = k2 [C] [D]
At equilibrium,
k1 [A] [B] = k2 [C] [D]
[A] [B]
k2
= Keq(equilibrium constant)
=
[C] [D]
k1
Law of mass action - for reversible reactions
(Effects of [S]and [P] on the direction in net reaction proceeds)
Background
[A] [B]
K eq=
[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 the 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
Catalysis
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: Introduction
Biocatalysts
Neither consumed / permanently altered
Colloidal organic compounds – proteins
Formed by living organisms
High specificity for their substrates and
reaction types
Enzymes: Introduction
No formation of unnecessary by-products
Function in dilute aqueous solutions under
mild conditions of temperature and pH
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
Ex: Lovastatin for HMG CoA reductase
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)
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 of enzymes contain sites for
binding cofactors or coenzymes
exists d/t tertiary structure of protein loss of
native enzyme structure derangement of
active site loss of function
For catalysis to take place substrate/s should
bind to the substrate binding site reversibly by
weak non-covalent bonds (Hydrogen bond, Van der
walls force, hydrophobic interactions)
Active site
Not rigid in structure and shape
Flexible to promote the 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 the 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 the enzymes
act to catalyze the reaction
Enzymes are much larger than the substrates
they act on
Holoenzyme, Apoenzyme
Some enzymes contain a non
prosthetic group other than
component
protein
protein
Holoenzyme=Apoenzyme+cofactor
(protein) (non protein)
Cofactors
Non-protein factors required for catalysis
Bind to the catalytic site
organic or inorganic
Organic cofactors
-prosthetic groups -- tightly bound
-coenzymes – released from active site
during reaction
Inorganic cofactors
– activators
Cofactors
coenzymes
organic
cofactors
inorganic
Prosthetic
group
activators
Exceptions -- FMN ,FAD, and biotin are tightly
bound to enzymes, but called as coenzymes
Prosthetic group
tightly bound to enzyme by covalent bond
cannot be separated from enzyme by Dialysis
Ex
-Biotin in carboxylases
Apoenzyme + Prosthetic group = Holoenzyme
(active enzyme)
Coenzymes
derivatives of vitamins
Bound reversibly by weak non-covalent bonds
to active site and released during the reaction
separated easily from enzyme by dialysis
Affinity for the enzyme is similar to substrate
chemically changed by catalysis
considered as co-substrate
Function: carriers of various groups
Coenzymes
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)
NAD+
Niacin(Vit B2)
FAD and FMN
Riboflavin (Vit B3)
Hydroxy ethyl
Hydrogen/electron
Hydrogen/electron
Pyridoxal phosphate(PLP)
Coenzyme A (CoA)
Biotin
Folate coenzymes
Adenosine tri phosphate(ATP)
Amino group
Acyl group
CO2
One carbon group
Phosphate
Pyridoxine(VitB6)
Pantothenic acid
Biotin
Folic acid
-------
Inorganic Cofactor/Activators
Metals
2 types - Metal activated enzyme
- Metallo enzymes
metal activated enzymes
metal is not tightly bound by the enzyme
Ex *ATPase (Mg2+)
*Enolase(Mg2+)
*Chloride (Cl-) -salivary amylase
*Ca2+ and Pancreatic lipase
Inorganic Cofactor/Activators
Metallo enzymes
Metals are tightly bound with enzymes
Ex - *alcohol dehydrogenase (zinc)
*carbonic anhydrase (zinc)
*DNA Polymerase (zinc)
*Xanthine oxidase(molybdenum)
*Catalase, peroxidase(Iron)
*Cytochrome oxidase(iron)/(copper)
*Hexokinase,
pyruvate
kinase(Mg2+)
*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 diagram
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
Michaelis- Menten Theory
Enzyme E combines with a single substrate S
to form Enzyme-Substrate 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
Fischer’s
Lock and Key Model
Fischer’s
Lock and Key Model
Theory active site of enzyme is pre-shaped
& rigid
Is complimentary to the substrate
Fit exactly into one another like key in lock
Explains only enzyme specificity
 Fails to explain the flexibility shown by
allosteric enzymes
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
Model:
*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”
Ex:
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
Classification of enzymes
The International Union of Biochemistry and
Molecular Biology (IUBMB)
6 major classes of enzymes
Mnemonic: OTHLIL
Oh Thank Heaven Learning Is Lively
Classification of enzymes
1.
2.
3.
4.
Oxidoreductases oxidation-reduction
Transferases transfer of group of atoms
Hydrolases hydrolysis
Lyases cleavage of bonds without
hydrolysis
5. Isomerases rearrangement of atoms
6. Ligases joining of molecules (using ATP)
Oxidoreductases
Catalyzes oxidation of one substrate with
simultaneous reduction of another substrate
or coenzyme
Alcohol dehydrogenase
Alcohol
NAD+
Aldehyde
NADH +H+
Malate dehydrogenase
Malate
NAD+
Oxaloacetate
NADH +H+
Oxidoreductases
AH2 + B
A + BH2
Transferases
These enzymes catalyze transfer of a group
other than H such as- amino, phosphoryl,
methyl, from one substrate to another
A- X+ B
A + B- X
Transferases
Transfer groups from one substrate to
another
Ex
Hexokinase
Hexose
ATP
Hexose-6-phosphate
ADP
Alanine transaminase
Pyruvate
Glutamate
Alanine
α-ketoglutarate
Hydrolases
These enzymes catalyze cleavage of a
molecule by addition of water (hydrolysis)
cleaves ester, peptide, glycosidic bonds
A–B + H2O
A-OH + B–H
Hydrolases
Ex
Lactase
Sucrase
Lactase
Lactose + H2O
Glucose + Galactose
Sucrase
Sucrose + H2O
Glucose + Fructose
Lyases
break bonds by other than hydrolysis
Fructose 1, 6 bis phosphate
Aldolase
Dihydroxyacetone
phosphate
Glyceraldehyde-3phosphate
Fumarase
Malate
Fumerate + H2O
Isomerases
These enzymes
substrates.
produce
isomers
of
Include racemases, epimerases and cis-trans
isomerases.
A
A'
Isomerases
Phosphohexose isomerase
Glucose 6-P
Fructose P
Phosphotriose isomerase
Glyceraldehyde 3-P
Dihydroxyacetone 3-P
Ligases
These enzymes catalyze synthetic reactions
two molecules joined by covalent bond at the
expenses of ‘high energy phosphate bond’ of,
usually ATP hydrolysis of ATP
Synthetases
A + B
C
ATP
ADP + Pi
Ligases
Pyruvate carboxylase
Pyruvate + CO2
ATP Biotin
Oxaloacetate
ADP + Pi
Acetyl CoA carboxylase
Acetyl CoA + CO2
ATP
Malonyl CoA
Biotin ADP + Pi
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-P + 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
Eg:
-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
The rate of reaction catalyzed by the enzyme
is proportionate to the quantity of enzyme
present
International unit (I.U)
Katal
Turnover number
Enzyme units
Katal
The number of mol of substrate transformed
per second per liter of sample.
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
Enzyme units
Turnover number
The 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
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
substrate specificity of enzymes
explains
Enzyme specificity : Types
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 : Types
Bond Specificity
Act on substrate having specific bonds
All proteolytic enzymes
Ex:
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 : Types
Group Specificity
Act on substrate having specific groups
Act on a structurally related substances
Ex:
Hexokinase - several hexoses to form the
respective hexose-6-phosphate
Enzyme specificity : Types
Stereo specificity
Act on only one type of isomers (exceptionisomerase)
Humans enzymes are specific for D–
carbohydrates and L-aminoacids
Ex:
L amino acid oxidase
D amino acid oxidase
Enzyme kinetics
Study of all the factors that affect the rates of
enzyme catalyzed reactions
The study of rate of enzyme catalyzed
reactions & factors affecting these reaction
rates
Enzyme kinetics
Enzyme concentration
Substrate concentration
Temperature
pH
Product concentration
Presence of coenzymes,
inhibitors
activators
or
Importance of studying
Enzyme kinetics
understanding mechanism of enzyme action
understanding
inhibition
mechanism
of
clinical diagnosis (measuring
enzymes in disease conditions )
Understanding disease processes
enzyme
activity
of
Basics
catalyst is required in much
concentration than the substrates
Similarly in an enzyme catalysed reaction
[E] <<< [S]
[E] nmoles/liter and [S] mmol/liter
lower
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
½ Vmax------------- B
Velocity
A
x
Km Substrate concentration
Effect of Substrate Concentration
Vmax
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 and Glucokinase
Hexokinase is present in all cells except
hepatocytes and β-cells of pancreas
Glucokinase is present in hepatocytes and βcells
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 and Glucokinase
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
Graph: 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
Effect of temperature
on enzyme activity
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
Graph: 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