Download Enzyme Activity

Enzymes:
Nothing works without enzymes!
• How important are enzymes?
– all chemical reactions in living organisms
require enzymes to work
enzyme
• building molecules
– synthesis enzymes
• breaking down molecules
We can’t live
without enzymes!
– digestive enzymes
– enzymes speed up reactions
• “catalysts”
+
enzyme
+
Enzymes aren’t used up
• Enzymes are not changed by the reaction
– used only temporarily
– re-used again for the same reaction with other
molecules
substrate
active site
product
enzyme
• Most enzymes are Proteins
(tertiary
and
quaternary
structures)
• Act as Catalyst to accelerates
a reaction
• Are specific
for what they
will catalyze
It’s shape that matters!
• Lock & Key model
– shape of protein
allows enzyme &
substrate to fit
– specific enzyme for
each specific
reaction
2
1
3
The Lock and Key Hypothèses
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Fit between the substrate and the active site of the enzyme is
exact
Like a key fits into a lock
Temporary structure called the enzyme-substrate complex
formed
Products have a different shape from the substrate
Once formed, they are released from the active site
Leaving it free to become attached to another substrate
The Lock and Key Hypothesis
S
E
E
E
Enzymesubstrate
complex
P
P
© 2007 Paul Billiet ODWS
Reaction coordinate
Enzyme may be
used again
Enzyme Kinetics Equation
The active site
• One part of an enzyme, the active site, is particularly
important
• The shape and the chemical environment inside the
active site to proceed more easily
© H.PELLETIER, M.R.SAWAYA
ProNuC Database
Chemical reactions

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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 (ES)
Making réactions go faster

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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.
Factors affecting Enzymes

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substrate concentration
pH
temperature
inhibitors
1- 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

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Many are a lot lower, cold water fish will die at 30°C
because their enzymes denature
A few bacteria have enzymes very high temperatures up to
100°C
Most enzymes however are fully denatured at 70°C
Temperature
reaction rate
human
enzymes
37°
temperature
What’s
happening
here?!
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.
The effect of enzyme inhibition
Irreversible inhibitors: Combine with the
functional groups of the amino acids in the active
site, irreversibly.
Examples: nerve gases and pesticides, containing
combine with serine residues in the enzyme.

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
2. 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.
• 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 decrease Vmax, but have no
effect on Km
Kompetitive Inhibition:
Km Increases; no change in Vmax.
Non-kompetitive inhibition:
No Km change, but Vmax decreases
Applications of inhibitors
• Negative feedback: end point or end product
inhibition
• Poisons snake bite, nerve gases.
• Medicine antibiotics
© 2007 Paul Billiet ODWS
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
ALP
• Alkaline phosphatase
• The enzymes phosphatase catalyze hydrolysis of
phosphate esters to free inorganic phosphate
• The measurement of ALP activity in vitro is
based on artificial substrate used
• The artificial substrate p-nitrophenylphosphate
• the enzyme will hydrolysis it to
P-nitrophenol +phosphate ion
• P-nitrophenol is bright yellow and other
reactant and product are colorless /; thus
formation of product can measurement by
specphotometer 400nm
• The intensity of color indicator how much
enzyme acted on substrate
ALP is present in most human tissues
•
•
•
•
•
Bone
Liver
Spleen
Kidney
Intestine
• Diagnostic of ALP significance :
1. Hepatic disorder
2. Rickets
3. Paget disease
For enzymes…
What matters?
SHAPE!