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Enzymes
Introduction
 Kinetic reactions:
• all chemical reactions in living organisms require enzymes to
work
For building , breaking down molecules or speed up the
reactions.
 Enzymes are :a biological catalyst. Usually a globular protein
molecule produced by living organisms that can speed up a
specific chemical reaction without itself being destroyed or
changed in any way.
 They are produced by the living organism and are usually
present in only very small amounts in various cells.
Enzymes properties
 Enzymes aren’t used up
 Enzymes are not changed by the reaction
• re-used again for the same reaction with other molecules.
 Most enzymes are Proteins (tertiary and quaternary
structures)
 Act as Catalyst to accelerates a reaction
 Are specific for what they will catalyze
Enzyme Kinetics Equation
The Lock and Key Hypothesis
S
E
E
E
Enzyme may be used
again
Enzyme-substrate
complex
P
P
Reaction coordinate
© 2007 Paul Billiet ODWS
Making réactions 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.


Enzymes can increase the rate of reactions without increasing the temperature.
They do this by lowering the activation energy.
Factors affecting Enzymes




substrate concentration
pH
temperature
inhibitors
Substrate concentration:
Reaction
velocity
Substrate concentration
 The increase in velocity is proportional to the substrate
concentration
© 2007 Paul Billiet ODWS
Substrate concentration:
Vmax
Reaction
velocity
Substrate concentration
 Faster reaction but it reaches a saturation point when all the enzyme
molecules are occupied. Vmax
© 2007 Paul Billiet ODWS
PH
 Extreme pH levels will produce denaturation
 most human enzymes = pH 6-8
 depends on where in body
 pepsin (stomach) = pH 3
 trypsin (small intestines) = pH 8
 The structure of the enzyme is changed
 The active site is distorted and the substrate molecules
will no longer fit in it
Enzyme
activity
Trypsin
Pepsin
1
3
5
7
pH
9
11
Temperature
 Effect on rates of enzyme activity
 Optimum temperature
 human enzymes
 35°- 40°C (body temp = 37°C)
 Raise temperature (boiling)
 denature protein = unfold = lose shape
 Lower temperature T°
 molecules move slower
 fewer collisions between enzyme & substrate
Temperture and Enzymes ACTIVITY in
human
 Michaelis and menten at low substrate concentrations, the
enzyme is not saturated with the substrate and the reaction is
not proceeding at maximum velocity whereas when the
enzyme is saturated with substrate, maximum velocity is
observed.
Inhibitors
Inhibitors are chemicals that reduce the rate of enzymatic
reactions.
 They are usually specific and they work at
low
concentrations.
 They block the enzyme but they do not usually destroy it.
 Irreversible inhibitors:
 Combine with the functional groups of the amino acids in the
active site, irreversibly.

Reversible inhibitors:
There are two categories

1.
•
•
•
Competitive:
These compete with the
substrate molecules for the
active site.
The inhibitor’s action is
proportional
to
its
concentration.
Come over these problem by
adding more substrate
 Km : The addition of a competitive inhibitor increases the
observed Km for a given substrate.
 Therefore, in the presence of a competitive inhibitor, more substrate is
needed to achieve
 Vmax: Competitive inhibitors do not alter Vmax.
 The effect of a competitive inhibitor is reversed by increasing [S].
 high substrate concentration, the reaction velocity reaches the same
Vmax as that observed in the absence of the inhibitor.
 This is because at the higher concentration the active site will be
saturated with substrate which means the inhibitor cannot bind
Non-competitive: (allosteric effect)
 These are not treated 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.
2.
 Km : Non-competitive inhibitors do not interfere with the binding of




substrate to enzyme.
Thus, the enzyme shows the same Km in the presence or absence of the
non-competitive inhibitor.
Vmax: Increasing the concentration of substrate does not overcome
non-competitive inhibition.
Non-competitive inhibitors therefore decrease the Vmax of the
reaction.
Non-competitive inhibitors therefore simply reduce the amount of
active enzyme so they decreaseVmax, but have no effect on Km
Kompetitive Inhibition:
Km Increases; no change inVmax.
Non-kompetitive inhibition:
No Km change, but Vmax decreases
Michaelis-Menten Equation
Glossary
 Active site: The region of an enzyme molecule which binds the
substrate and carries out the catalytic reaction
 Enzyme : A biological catalyst. Usually a globular protein molecule
produced by living organisms that can speed up a specific chemical
reaction without itself being destroyed or changed in any way.
 K m: (Michaelis constant) The substrate concentration at which an
enzyme catalysed reaction proceeds at half the maximum velocity.
 V max: (Maximum velocity) The maximum initial velocity of an
enzyme catalysed reaction; determined by increasing the substrate [S]
until a constant rate of product formation is achieved (i.e. saturating substrate
levels).
 A CATALYST is anything that speeds up a chemical
reaction that is occurring slowly
ALKALINE PHOSPHATASE
 Is a member of the phosphomonoestrases group. Which are
highly specific and act an only one substrate, alkaline
phosphatase has a broad substrate specificity and is named
alkaline phosphatase because its ph optimum is usually
around 9 but other broad spectrum phosphoesterases with ph
optima less than 7 are termed acid phosphatases.
Determination ALP in vitro
 Alkaline phosphatase activity by following the rate of
formation of products.
 ALP catalyzes in alkaline medium the transfer of the
phosphate group from 4 nitrophenylphosphate to 2 amino 2
methyle propanol, librating 4 nitrophenol
 The catalytic concentration is determined from the rate of 4
nitrophenol formation, measured at 400nm.
 The 4 nitrophenol is bright yellow but other reactants and
products are colorless in aqueous solution.
 The measurement of ALP
activity in vitro is based on
artificial substrate p-nitrophenylphosphate.
 The intensity of yellow color in the reaction solution thus
indicates the degree to which enzyme has acted upon
substrate.(how much enzyme acted on substrate).
Study enzyme kinetics
 Enzyme kinetics: the study of the rate at which an enzyme works.
 To examine it, when the substrate available to the enzyme one
would do the following:
1.
set up the series of tubes containing graded concentrations
of substrate
2. At time zero, a fixed amount of the enzyme preparation is added
3. Over the next few minutes, the concentration of product
formed, is measured. If the product absorbs light, we can easily
do this in a spectrophotometer
4. Early in the run, when the amount of substrate is in substantial
excess to the amount of enzyme, the rate we observed is the
initial of velocity Vi.
Experiment
 Facts..
 An assay is necessary to study an enzyme
 The assay is a measurement of a chemical reaction, which
might involve measuring the formation of the product (or
otherwise the decrease in substrate conc.)
Reagents and instruments
 18 labeled plastic tubes
 Micropipette
 Spectrophotometer
 ALP enzyme kit (ready to use)
 Blood serum
 5 N NaOH solution
Procedure
 Take 18 clean plastic tubes and label them from 1 to 18. Another
tube will be used as a blank. This will contain all the reagents
except of the enzyme.
 Make the substrate and buffer concentrations as described in the
given table (make sure to keep the total volume of all tubes stable
at 1.9 ml).
 Transfer 100 µl of serum to each tube.
 Mix substrate and serum solutions and incubate at 37c for 50
seconds.
 Add 0.5 ml of 5 N NaOH in each tube to stop the reaction.
 Read the absorbance at 400 nm.
 Plot reaction rate (Vi) on Y axis against substrate concentration [S]
on X axis.
Buffer
Substrate
Enzyme
5 N NaOH
μl
μl
μl
μl
1
100
1800
100
500
2
200
1700
100
500
3
300
1600
100
500
4
400
1500
100
500
5
500
1400
100
500
6
600
1300
100
500
7
700
1200
100
500
8
800
1100
100
500
9
900
1000
100
500
10
1000
900
100
500
11
1100
800
100
500
12
1200
700
100
500
13
1300
600
100
500
14
1400
500
100
500
15
1500
400
100
500
16
1600
300
100
500
17
1700
200
100
500
18
1800
100
100
500
Tube
Abs. at
400nm