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1. 3. Enzymes
And the Kinetic Affinity

3. What are Enzymes
 Biological Catalyst
 Specific a certain substrate by its R group
 Globular protein – water soluble
 Remain unchanged after the reactions
 Enzymes can break and bond!
 Nearly all metabolic reaction are enzymes-catalyzed
 Enzymes reduce activation energy – increase rate constant
RATE = K[A]*[B}y

4. Activation Energy
 All metabolic reaction needs extra activation energy – or
they don’t happen at all
 This can be provided in heating eg. What we did in benedict
test
 To change substrate to product, a brief raise in energy is
required – the amount is called the activation energy
 Change in shape of the product lowers the activation energy
 Enzymes can conduct even in lower temperature ie. High
temperature not needed.

5. Activation Energy

6. Intracellurlar/
Extracellular
 Intracellular: Used inside the cell – eg. ATPase,
helicase, polymerase
 Extracellular: Those secreted out of the cell eg.
Pancreatic enzymes – protease, amylase,
maltase, lipase

7. The Lock and key Theory
1. The enzyme has a cleft/ depression called the active site
2. Active site and the specific substrate has complementary
shape
3. The substrate meets the enzymes by random movement
4. The substrates fits into the cleft
5. The R group binds with the substrate
6. An enzyme-substrate complex is formed
7. The enzyme catalyzes the reaction – breaking it apart or
joining

8. Lock and key theory

9. The Induced Fit Theory
 Like the Lock-and-key
 Only here, it is recognized the enzyme is more
flexible – able to change shape slightly to fit the
substrate

10. pH effects on Enzymes
 Change of pH can disturb the ionic bond
which is important to the tertiary
structure of a proteins.
 Can also change the charges of the amino
acid – more hydrogen = more acidic
 pH measures the conc. of H+ ions - higher
conc. will give a lower pH

11. Increase in temperature
 Molecular movements speed up – more
random movements – more activities
 37 degrees – the optimum for bodily
enzymes
 Until up to 40 degree Celsius, all is good,
rate of reaction proportional to temp. –
where the hydrogen bond breaks -
denature

12. Decrease in temperature
 Less active enzymes
 However – this do not denature the
enzymes
 Certain animals can work with this
(Psychrophilic like cold, thermophiles
like it hot, hyperthermophils can not
grow anywhere lower than 70 degree
Celsius)

13. Course of an Enzyme
reaction
 Usually starts out quickly before going out on
a gentler curve
 At first every enzyme is paired up – this rate
depends on how quickly an enzyme can
catalyze , then release – this IS the RATE.
 Because after this point, the measure is
influenced by the amount of substrate left
although the rate is supposed to only
measure how fast an enzyme work.

14. Course of an Enzyme
reaction
 Imagine the enzyme as a factory worker
 You want to measure how fast S/he can finish the
work
 Now, you have a 50 toys that you want him/her to
piece together (the Substrate)
 But also imagine – as in a cell – you didn’t stack up the
toys on her desk, you leave them all over the room
 At the beginning, s/he’s quick to find the toys – in fact
s/he’ll randomly bump into those ones lying around

15. Course of an Enzyme
reaction
 So if you time at the beginning, you’ll
actually get the speed of her work
 But after she’s done, say, 25 of them. The
other 25 are hidden very well. Now she
has to look around for them.
 So if you time her now, you won’t actually
get the sped of her work – you will get the
speed of her looking for things.
 This applies similarly to enzymes

16. Course of an Enzyme
action
 At the beginning of the reactions, there are
enough substrates for the enzymes to work
with – so they’re working at the real speed
 Soon there are fewer substrates – enzymes
are waiting to be filled up – soon it stops
 Therefore the first 30 seconds usually gives
us The initial rate of reaction.

17. Enzyme Kinetics

18. Initial Rate vs. Substrate
 This graph is shown on page 200 – 201
 It plots the initial rate of reaction for each
substrate concentration – supposed to
show at which substrate concentration
the graph flattens out eg. Reaches Vmax
 INITIAL RATE OF REACTION IS THE
THEORETICAL VELOCITY OF A REACTING
ENZYME FOR EACH CONDITION

19. Steps to doing this
 First – understand our objective – we want
to find the maximum speed an enzyme
could work – to do that, we have to
increase its concentration to a point
where the enzyme is working so hard, it
can’t go any faster.
 That is our Vmax

20. Steps to doing this
 Back to the factory worker analogy.
 Now we want to know the fastest speed at
which s/he can work – not the normal
speed, the fastest one
 So what we do is we keep increasing the
amount of toys we want her to piece
together – measuring the initial rate of
work for everyone of them, because
remember? That’s the accurate rate when
she doesn’t have to go out to find toys

21. Steps to doing this
 In real world scenario, we make a range of
substrate concentration – 5%, 20%, 40%... whatever
 In the analogy, we have a range of toy numbers – 3,
7, 13, 17… whatever
 With enzymes, we measure the initial rate of
reaction for everyone of the set-up
 With the toys, we measure how fast it takes
him/her to work with 3, then measure how fast for
7, then 13 and so on and so forth

22. Steps to doing this
 What we expect…
 Enzymes with higher substrate concentration
would work faster
 When the factory worker works with 3 toy, s/he’s
gonna go very slow – but if there 15 toys lined,
s/he’ll be working at mad speed
 So when the substrate concentration is REALLY
HIGH, the enzyme will be working incredibly hard

23. As substrate concentration
(number of toys increases), the
initial rate of reaction (the
speed of worker’s work)
increases…
Until there are so many things
to do… the worker/enzymes
can not be any faster

24. Michaelis Menten Model
 The Michaelis Menten constant is used to
compare affinity of different enzymes to their
substrates.
 When the Enzymes are working at the hardest,
and they can not go any faster – we call this
Enzyme saturation
 This is the Vmax – a maximum rate in which an
enzyme can work at.
 All active sites of the enzyme are occupied

25. Vmax
 The Theoretical maximum velocity (speed) of
rate of activity of the enzyme before substrate
concentration becomes the limiting factor.
 Measured at the point of saturation – every
enzyme has a substrate (or all the enzymes
active sites are occupied).
 Measured by increasing substrate
concentration while leaving the enzyme
concentration constant

26. Km
 Substrate concentration at which the rate of
the enzyme activity is Vmax/2
 Km measures the affinity/ efficiency of an
enzyme – how quickly an enzyme reaches Vmax
 It only points to when a substrate is already
bound to an enzyme
 Kinda like acceleration – how quickly it
reaches the maximum speed.

28. Enzymes Inhibitors
 Competitive inhibitors: Bind at the active site of
an enzyme – thus competing with the substrate
 Non-competitive: Bind at another site other than
the active site

29. Inhibitions
 Competitive inhibition: When a substance
reduces the rate of activity of the enzyme
by competing with the substrate in
binding with the enzyme’s active site.
Increasing the concentration of the
substrate can reduce the degree of
inhibition
 Non-competitive inhibition: When a
substance reduces the rate of activity of
an enzyme, but increasing the
concentration of substrate does not
reduce the degree of inhibition. Such

30. Competitive
 Reduces Enzymes affinity – as it prevents the substrate
from joining with the enzymes
 Km increases (don’t forget Km is simply acceleration
expressed in the terms of distance[sub conc.] hence it
is inversely proportional to the enzyme affinity)
 Vmax doesn’t change because adding substrate can still
over come the effect
 If we add high enough Substrate concentration – they
can overtake inhibitor – and Vmax can still be reached

31. Non - Competitive
 Changes the enzymes conformation
 Can have both bounded at the same time
(Enzyme-Substrate-Inhibitor can form but
the enzymes do not work)
 No reduced affinity – Km stays the same
 However since products can not be
produced – Vmax decreases

32. Inhibitors roles
 Slow down rate of reaction eg. High temperature
 Big issues with inhibitors: If one swallows
methanol, it inhibits dehydrogenase – the
original substrate is given in large doses to
revers the effect.
 Irreversible inhibition – chemical permanently
binds or denature the enzymes eg. Nerve gas –
penicillin sometimes used to permanently block
bacterium pathways
 End product inhibition eg. When reaction has to
stop – end products accumulate to stop reaction

33. Immobilizing Enzymes
 Enzymes is immobilized for commercial purpose
 Lactase is used with milk to produce lactose-free
milk
 Lactase mixed with sodium alginate – then each
droplet put into calcium chloride – which then
immediately forms beads.
 These beads are arranged and milk is poured
through it .
 Advantages: Do not need to separate enzymes –
milk is not contaminated – lactase is not lost –
more tolerant to pH and temperature changes –