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Enzymes
Enzymes are PROTEINS
and certain class of RNA (ribozymes)
which ENHANCE
the rate of
THERMODYNAMICALLY
feasible REACTION and are
NOT PERMANENTLY ALTERED
IN THE PROCESS
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.
Reaction pathway
© 2007 Paul Billiet ODWS
Making reactions go faster
 Temperature increase molecules movement
 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
An enzyme controlled pathway
Enzyme controlled reactions proceed 108 to 1011 times faster
than corresponding non-enzymic reactions.
© 2007 Paul Billiet ODWS
The Enzyme Reaction
 Conventionally we say the enzyme acts on the
substrate (S) to yield products (P)
E
S→P
 Since E is a catalyst it remains unchanged at the
end of the reaction
Each reaction has a transition state where the substrate
is in an unstable, short-lived chemical/structural state.
Free Energy of
Activation
is symbolized by ΔG‡.
Enzymes act by
lowering the free
energy of the
transition state
Enzymes speed up metabolic
reactions by lowering energy barriers
 Enzyme speed reactions by lowering EA.
The transition state can be reached at
moderate temperatures.
 Enzymes do not change delta G.
It speed-up reactions that would occur
eventually.
The Enzyme Reaction
 Enzymes lower the free energy of activation
by binding the transition state of the
reaction better than the substrate
 The enzyme must bind the substrate in the
CORRECT ORIENTATION otherwise
there would be NO REACTION
 Not a lock & key but induced fit theory
 the enzyme and/or the substrate distort
towards the transition state
Biochemistry, Stryer, 5th edition
The Lock and Key Hypothesis
 Fit between the SUBSTRATE and
the ACTIVE SITE of the enzyme
 Same as KEY FITS INTO A LOCK
very precisely
© 2007 Paul Billiet ODWS
The Lock and Key Hypothesis (cont.,)
 Temporary structure called the enzymesubstrate complex formed
 PRODUCTS have a different shape from
the SUBSTRATE
 Once formed, PRODUCTS are released
from the active site
 Leaving it free to become attached to
another substrate
© 2007 Paul Billiet ODWS
The Lock and Key Hypothesis (cont.,)
Figure 8.9. Lock-and-Key Model of Enzyme-Substrate Binding.
In this model, the active site of the unbound enzyme
is complementary in shape to the substrate.
Biochemistry, Stryer, 5th edition
The Induced Fit Hypothesis
 The enzyme changes shape on substrate
binding
 The active site forms a shape
complementary to the substrate
only after the substrate has been bound
 SUBSTRATE BINDING INDUCES A
CHANGE IN THE ENZYME’S
CONFORMATION
Induced-Fit Model of Enzyme-Substrate
Binding
Figure 8.10. Induced-Fit Model of Enzyme-Substrate Binding.
In this model, the enzyme changes shape on substrate
binding. The active site forms a shape complementary to the substrate
only after the substrate has been bound.
Biochemistry, Stryer, 5th edition
Enzyme structure
 Enzymes are proteins
in nature
 They have a globular
shape
 A complex 3-D
structure
Human pancreatic amylase
© 2007 Paul Billiet ODWS
© Dr. Anjuman Begum
The active site
 Is one part of the
enzyme and it is
particularly
IMPORTANT
 The SHAPE and the
CHEMICAL
ENVIRONMENT
inside the active site
© H.PELLETIER, M.R.SAWAYA
ProNuC Database
© 2007 Paul Billiet ODWS
permits a chemical
reaction to proceed
more easily
The active site (cont.,)
 Typically a pocket or groove on the
surface of the protein into which the
substrate fits
 The specificity of an enzyme
 fit between the active site and the substrate
 Enzyme changes shape
 tighter induced fit, bringing chemical groups
in position to catalyze the reaction
The active site is an enzyme’s catalytic
center
 In most cases substrates are held in the
active site by WEAK INTERACTIONS
 A single enzyme molecule can catalyze
thousands or more reactions a second
 Enzymes are UNAFFECTED by the
reaction and are REUSABLE
Enzymes can be classified according
to the chemical reactions they catalyze



1. Oxidoreductases: oxidation/reduction
(eg. DEHYDROGENASES)
2. Transferases: group transfer
(eg. KINASES)
3. Hydrolases: hydrolysis
(eg. PROTEASES)
Enzymes can be classified according
to the chemical reactions they catalyze



4. Lyases: lysis, generating double bond
(eg. SYNTHASES)
5. Isomerases: rearrangement
(eg. RACEMASES)
6. Ligases: ligation requiring ATP
(eg. SYNTHETASES)
Cofactors
 An additional
non-protein
molecule that is
needed by some
enzymes to help
the reaction
Nitrogenase enzyme with Fe, Mo and ADP
cofactors
© 2007 Paul Billiet ODWS
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 IMPLICATIONS FOR
SIGNAL TRANSDUCTION; NATURE 387:370 (1997)
Cofactors (cont.,)
 Tightly bound cofactors
are called PROSTHETIC
GROUPS
 Cofactors that are bound
and released easily are
called COENZYMES
 Many vitamins are
coenzymes
© 2007 Paul Billiet ODWS
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
IMPLICATIONS FOR SIGNAL TRANSDUCTION;
NATURE 387:370 (1997)
Common Coenzymes
The substrate
 SUBSTRATES are REACTANTS
activated by enzyme
 Enzymes are specific to their substrates
 The specificity is determined by the
active site
© 2007 Paul Billiet ODWS
Substrate orientation
Substrate specificity
Enzymes are substrate specific
 When a substrate(s) binds to an enzyme, the
enzyme catalyzes the conversion of the substrate
to the product.
– SUCRASE is an enzyme that binds to
sucrose and breaks the disaccharide
into fructose and glucose
Enzymes are substrate specific (cont.,)
 The rate that a specific number of enzymes
converts substrates to products depends in
part on substrate concentrations
 At low substrate concentrations, an increase
in substrate speeds binding to available
active sites
 – However, there is a limit to how fast a
reaction can occur
Enzymes are substrate specific (cont.,)
 At some substrate concentrations, the active
sites on all enzymes are engaged, called
ENZYME SATURATION
 The only way to increase productivity at
this point is to add more enzyme
molecules
Factors affecting Enzymes




substrate concentration
pH
temperature
inhibitors
© 2007 Paul Billiet ODWS
Substrate concentration:
Non-enzymic reactions
Reaction
velocity
Substrate concentration
 The increase in velocity is proportional to the
substrate concentration
© 2007 Paul Billiet ODWS
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
The effect of pH
Optimum pH values
Enzyme
activity
Trypsin
Pepsin
1
© 2007 Paul Billiet ODWS
3
5
7
pH
9
11
The effect of pH
 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
 Extreme pH levels will produce
DENATURATION OF ENZYME
 The structure of the enzyme is changed
 The active site is distorted and the substrate
molecules will no longer fit in it
© 2007 Paul Billiet ODWS
The effect of temperature
 LITTLE activity at LOW temperature
 Rate INCREASES with temperature
 Most active at optimum temperatures
(usually 37°C in humans)
 Enzyme-controlled reactions follow this
rule as they are chemical reactions
 BUT at high temperatures proteins
DENATURE
© 2007 Paul Billiet ODWS
The effect of temperature
Q10
Enzyme
activity
0
© 2007 Paul Billiet ODWS
10
20
30
40
Temperature / °C
Denaturation
50
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
Types of inhibitors
 Irreversible inhibitors:
 Combine with the functional groups of the
amino acids in the active site, irreversibly.
 Examples:
NERVE GASES and PESTICIDES,
containing organophosphorus, combine with
serine residues in the enzyme acetylcholine
esterase.
© 2007 Paul Billiet ODWS
Types of inhibitors (cont.)
 Reversible inhibitors:
 These can be washed out of the
solution of enzyme by dialysis.
 There are two categories
 COMPETITIVE
 NON-COMPETITIVE
© 2007 Paul Billiet ODWS
Types of inhibitors (cont.)

COMPETITIVE:

These compete with the
substrate molecules for
the active site.
The inhibitor’s action is
proportional to its
concentration.
Resembles the
substrate’s structure
closely.


© 2007 Paul Billiet ODWS
E+I
Reversible
reaction
EI
Enzyme inhibitor
complex
Types of inhibitors (cont.)


NON-COMPETITIVE:
These are not influenced by the concentration
of the substrate. It inhibits by binding
irreversibly to the enzyme but not at the active
site.
 Examples
 Cyanide combines with the Iron in the
enzymes cytochrome oxidase.
 Heavy metals, Ag or Hg, combine with –SH
groups.
© 2007 Paul Billiet ODWS
Enzymes are used in biological
washing powders
 PROTEASES break down the coloured,
insoluble proteins that cause stains to
smaller, colourless soluble polypeptides.
 LIPASES can hydrolyze lipid spots at
lower temperatures
Enzymes are used in the food industry
 PECTINASE
break down substances
in apple cell walls and
enable greater juice
extraction.
Enzymes are used in the food industry
 LACTASE
breaks down lactose in milk into
glucose and galactose.
This makes milk drinkable for lactose
intolerant people.