Enzymes are biological catalysts. They play some of the most important roles in the processes of life sustenance. They are presence even at the tiniest level of metabolism - acting as the lubricant for life to progress smoothly. Without enzymes, complex life would not be possible.

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. An enzyme-product complex is formed
9. The product leaves the enzymes
10. The enzymes remain unchanged – ready to go for the next substrate

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 58 – 59
 It plots the initial rate of reaction for each substrate
concentration – supposed to show that, at which
substrate concentration does 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 objectie – 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
 When the Enzymes are working at the hardest, and
they can not go any faster - Enzyme saturation
 This is the Vmax – a maximum rate in which an
enzyme can work at.

25. Vmax
 The Theoretical maximum rate that an enzyme can
perform
 Measured at the point of saturation – every enzyme
has a substrate
 Measured by increasing substrate concentration
while leaving the enzyme concentration constant

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

28. The Double Reciprocal Plot
 In reality, the enzyme continues to work, so the rate increases
little by little and would only flatten out at infinity
 Because infinity is not on the graph – we can’t accurately read
off the Vmax - we can guess at best
 Solution: Since 1/infinity = 0 (if n tends to infinity but if an
infinity value is fixed, then it’s 1)
 Therefore, by plotting a graph of 1/(Substrate concentration)
against a graph of 1/ (velocity) – we receives a reciprocal
graph that at whichever point that it reaches the 0 substrate
concentration - the point when it touches the y axis (because
that is the infinity substrate concentration in reciprocal term) –
will be equal to 1/Vmax.
 -1/Km (because we can’t have negative Km) can be found at the
point of x-axis interception

30. Relationship in retrospect
 First: Rate of reaction at 30 seconds is INITIAL
RATE OF REACTION
 INITIAL RATE OF REATION is the VELOCITY in
Michaelis-Menten model which tries to calculate the
Vmax (a theoretical maximum velocity of a certain
enzyme) and how quickly the enzyme can reach
that, expressed in the terms of Km
 Km = ½ of Vmax – calculated by the reciprocal plot or
a hyperbolic normal plot

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

32. 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 sit.
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 inhibitors may
bind to other areas of the enzymes that are not
active sites

33. 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

34. Non - Competitive
 Change the enzymes formation
 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 product can not be produced – Vmax
decreases

36. 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 eg. When
maltose inhibits amylase

37. 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 – because held in beads
so structure are not easily changed, and the bead
formation protect the vulnerable parts.