ENZYMES

Protein catalysts that increase the rate of

reactions without being changed in the overall
Process.

© 2007 Paul Billiet ODWS

HISTORY OF ENZYMES
 Biological catalysis was first recognized &
described in late 1700.(digestion of meat by
secretion of stomach).
 In 1850, L. Pasteur found that sugar
conversion into alcohol is by ferments
 In1897, E.Buchnar found that yeast extract
can ferment sugar to alcohol.
 In1926, J.Summner isolated the Urease and
found that they are proteins.F.W.Kuhne called
 These compounds as ENZYMES(from Greek
 enzymos
 About 1930, J.B.S. Haldane suggested that
weak interactions might be used to catalyze a
reaction.This insight lies at heart of our
current understanding of enzymes catalysis.
Properties Of Enzymes
 Catalysts for biological reactions
 Most are proteins
 Lower the activation energy
 Increase the rate of reaction
 Activity lost if denatured
 May be simple proteins
 May contain cofactors such as metal ions or
organic (vitamins)
 Work selectively & efficiently
5
Reaction pathway

© 2007 Paul Billiet ODWS

Making reactions go faster
 Increasing the temperature make molecules move
faster
 Biological systems are very sensitive to temperature
changes.
 Enzymes can increase the rate of reactions without
increasing the temperature.
 They do this by lowering the activation energy.
 They create a new reaction pathway “a short cut”

© 2007 Paul Billiet ODWS

Chemical reactions
 Chemical reactions need an initial input of energy =
THE ACTIVATION ENERGY
 During this part of the reaction the molecules are
said to be in a transition state.
 E +S-----------EP---------E +P

© 2007 Paul Billiet ODWS

 An enzyme controlled pathway

 Enzyme controlled reactions proceed 108 to 1011 times faster

than corresponding non-enzymic reactions.
© 2007 Paul Billiet ODWS
 Enzyme Activity Unit

S → P mmole

Product [P]
t
vo = [P] / min Slope
tan

Unit = mmole/min 0 10 20 30 40
Reaction time (min)

Juang RH (2004) BCbasics

Specific Activity Units y
Activity = Protein (mg) y = tan
x x 10
 Turn Over Numbers of Enzymes

Enzymes Substrate kcat (s-1)

Catalase H2O2 40,000,000
Carbonic anhydrase HCO3- 400,000
Acetylcholinesterase Acetylcholine 140,000
b-Lactamase Benzylpenicillin 2,000
Fumarase Fumarate 800

The number of product transformed from substrate

by one enzyme molecule in one second
11

Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.263
Cofactors and
Coenzymes
Cofactors
 An additional non-
protein molecule that is
needed by some
enzymes to help the
reaction
 Tightly bound cofactors
are called prosthetic
groups
 Cofactors that are bound
and released easily are
called coenzymes
 Many vitamins are
coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors
Jmol from a RCSB PDB file © 2007 Steve Cook
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS
© 2007 Paul Billiet ODWS
IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)
Catalysis by Cofactors
 Inorganic ions are required by some enzymes
for their working are called as cofactors
Examples
 Cu2+ Cytochrome oxidase
 Fe2+,Fe3+ Cytochrome
oxidase,Catalase,Peroxidase
 Zn2+ Carbonic anhydrase
 Ni2+ Urease
 K+ Pyruvate kinase
Factors affecting Enzymes
 Enzyme & substrate concentration
 pH
 temperature
 inhibitors

© 2007 Paul Billiet ODWS

Enzyme Concentration and Reaction Rate
 The rate of reaction increases as enzyme concentration
increases (at constant substrate concentration)
 At higher enzyme concentrations, more enzymes are
available to catalyze the reaction (more reactions at once)
 There is a linear relationship between reaction rate and
enzyme concentration (at constant substrate concentration)
Substrate concentration: Enzymic reactions

Vmax

Reaction
velocity

Substrate concentration
 Faster reaction but it reaches a saturation point when all the
enzyme molecules are occupied.
 If you alter the concentration of the enzyme then Vmax will
change too.
© 2007 Paul Billiet ODWS
Substrate Concentration and
Reaction Rate
 The rate of reaction increases as substrate
concentration increases (at constant enzyme
concentration)
 Maximum activity occurs when the enzyme
is saturated (when all enzymes are binding
substrate)
 The relationship between reaction rate and
substrate concentration is exponential, and
asymptotes (levels off) when the enzyme is
saturated
The effect of pH
 Extreme pH levels will produce denaturation
 The structure of the enzyme is changed
 The active site is distorted and the substrate
molecules will no longer fit in it
 At pH values slightly different from the enzyme’s
optimum value, small changes in the charges of the
enzyme and it’s substrate molecules will occur
 This change in ionisation will affect the binding of
the substrate with the active site.
© 2007 Paul Billiet ODWS
Factors Affecting Enzyme Action

Reaction
Rate
Optimum pH

3 5 7 9 11

pH
22
pH profiles of enzymatic reactions
Amylase

Pepsin

UCI Bio199 Independent Research

The effect of temperature
 For most enzymes the optimum temperature is about
30°C
 Many are a lot lower,
cold water fish will die at 30°C because their
enzymes denature
 A few bacteria have enzymes that can withstand very
high temperatures up to 100°C
 Most enzymes however are fully denatured at 70°C

© 2007 Paul Billiet ODWS

Factors Affecting Enzyme Action
Optimum temperature

Reaction
Rate

Low High
Temperature
25
The effect of temperature

Q10 Denaturation
Enzyme
activity

0 10 20 30 40 50
Temperature / °C

© 2007 Paul Billiet ODWS

The effect of temperature
 Q10 (the temperature coefficient) = the increase in
reaction rate with a 10°C rise in temperature.
 For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with every
10°C rise in temperature)
 Enzyme-controlled reactions follow this rule as they
are chemical reactions
 BUT at high temperatures proteins denature
 The optimum temperature for an enzyme controlled
reaction will be a balance between the Q10 and
denaturation.
© 2007 Paul Billiet ODWS
Inhibitors
 Inhibitors are chemicals that reduce the rate of
enzymic reactions.
 The are usually specific and they work at low
concentrations.
 They block the enzyme but they do not
usually destroy it.
 Many drugs and poisons are inhibitors of
enzymes in the nervous system.
© 2007 Paul Billiet ODWS
The effect of enzyme inhibition
1. Competitive: These
compete with the
substrate molecules for E+I EI
the active site.
Reversible Enzyme inhibitor
The inhibitor’s action is reaction complex
proportional to its
concentration.
Resembles the substrate’s
structure closely.

© 2007 Paul Billiet ODWS

Competitive Inhibition
A competitive inhibitor
 Has a structure similar to substrate

 Occupies active site

 Competes with substrate for active

site
 Has effect reversed by increasing
substrate concentration

30
The effect of enzyme inhibition

Succinate Fumarate + 2H++ 2e-

Succinate dehydrogenase

CH2COOH COOH CHCOOH

CH2

CH2COOH CHCOOH
COOH
Malonate

© 2007 Paul Billiet ODWS

examples
 Ritomir, HIV Protease Inhibitor(viral
replication)
 Captopril, Angiotension Converting
Inhibitor(ACE)
 Sulfonamides, Folic Acid inhibitor in
bacterial cells
 Fluorouracil ,RNA synthesis Inhibitor

32
Noncompetitive Inhibition
A noncompetitive inhibitor
 Does not have a structure like substrate

 Binds to the enzyme but not active site

 Changes the shape of enzyme and active site

 Substrate cannot fit altered active site

 No reaction occurs

 Effect is not reversed by adding substrate

33
 EDTA ,Inhibits calcium activated proteases
and the blood coagulation pathway.
 Irreversible inhibitors ,Nerve gas and
 Melathion are inhibitors of acetylcholine
estrase and are termed as suicide inhibitors.

34
Applications of inhibitors
 Negative feedback: end point or end product
inhibition
 Poisons snake bite, plant alkaloids and nerve
gases.
 Medicine antibiotics, sulphonamides,
sedatives and stimulants

© 2007 Paul Billiet ODWS

Properties of enzymes
 Active Site it contains AAs side chains that creates 3D surface
complementary to the substrate
 Catalytic efficiency 10(3)-----10(12)
 Specificity a; highly specific
 b ; relative specific
 Holoenzyme it refers to the active enzyme with its non protien
component if it is a metal ion it is called a cofactor if it is a small organic
molecule it is called COENZYME
 REGULATION Enzymes activity can be regulated , it can be activated
OR inhibeted
 ISOZYMES same funtion but different composition, found in different
tissues

36
Properties of Enzymes
 The direction of the enzyme reaction: Most
enzyme reaction are reversible,i.e.the same enzyme
can catalyze reactions in both directions but the
actual direction of reactions depends upon certain
factors e.g. availability of free energy.
 Proenzymes or zymogens: many enzymes are
inactive when first produced; later by the action of
another enzyme or some other substance these are
converted to their active form.

37
Proenzymes or Zymogens
 Pepsinogen is the zymogen form pepsin and
activated by gastric HCl
 Autocatalytic activation of pepsinogen by its own
active form.
 Pepsinogen contains 363 amino acids loses 42 amino
acids to form trypsin
 Trypsinogen contain 229 amino acids and loses 6
amino acid to form trypsin

38
Regulatory ENZYMES
 The activity of enzymes is regulated by several
means.
 Irreversible covalent modification such as in
pepsin activation
 Reversible covalent modification,such as
Phosphorylation and dephosphorylation of certain
enzymes of Glycogen metabolism i,e. Glycogen
Phosphorylase b is converted to
 Glycogen phosphorylase a by phosphorylation
 Glycogen Synthase.(deactivated)
 Allosteric Modulation Inhibition and activation
39
 Allosteric Regulation(other site )
 Non covalent interaction of modifiers with
enzyme Glycogen Synthase.
 Effectors , cAMP,cGMP,Inositol 1,4,5-
triphosphate,1,2-diacyglycerol.
 Inhibitors Homotropic effect(substrate)
 ,Heterotrophic effect(non substrate)
 Feed back inhibition
Ca ion with Calmodulin
modification

 Ca ion with Calmodulin modification

 Calmodulin a protein by taking calcium ion
 Is modified and can activate and inhibit many
enzymes. adenylyl cyclase, phosphorylase
kinase,nitric oxide synthase etc.
Classification of Enzymes
 Enzymes are classified according to the type of reaction
they catalyze:

Class Reactions catalyzed

 Oxidoreductases Oxidation-reduction
 Transferases Transfer groups of atoms
 Hydrolases Hydrolysis
 Lyases Add atoms/remove atoms
to/from a double bond
 Isomerases Rearrange atoms
 Ligases Use ATP to combine
molecules
Classification of Enzymes
 Enzymes are classified according to the type of reaction
they catalyze:

Class Reactions catalyzed

 Oxidoreductases Oxidation-reduction
 Transferases Transfer groups of atoms
 Hydrolases Hydrolysis
 Lyases Add atoms/remove atoms
to/from a double bond
 Isomerases Rearrange atoms
 Ligases Use ATP to combine
molecules
Classification of Enzymes
Class Reactions catalyzed
 Oxidoreductoases oxidation-reduction,A Hydrogen
or electron donor is one of the substrate
 Transferases transfer group of atoms,-X+B----
----A+B-X
 Hydrolases hydrolysis, cleavageof C-C,C-N,C-O
etc
 Lyases add/remove atoms (C-C,C-N,C-
O) to/from a double bond
 Isomerases rearrange atoms ,
 ligases combine molecules
using ATP 44
DETAIL OF SIX MAIN CLASSES
 oxidoreductases
 1; Anaerobic dehydrogenases
 utilize NAD OR NADP as
coenzyme
 Pyruvic acid+NADH2-----LDH-------------
LATIC ACID+NAD+
 Aerobic dehydrogenases use oxygenas H-
accepter
 Ascorbic Acid +1/2O2---Oxidases –
Dehydroascorbic acid

45
 Transferases Transmethylases
 Transacylases
 Transcarboxylases
 Hydrolases Lipases
 Carbohydrases
 Peptidases OR
 protienases
 Lyases Decarboxylases
 Deaminases

46
 Isomerases Mutases
 Epimerases
 Ligases catalyzing the bond
between two substrates
 Glutamine synthetase
 Pyruvate carboxylase

47
Lyases, Isomerases and Ligases
Diagnostic Enzymes
 The levels of diagnostic enzymes in the blood can be
used to determine the amount of damage in specific
tissues