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.
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.
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
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.
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.
27.
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
29.
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
35.
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.
