AS Level Biology - 3) Enzymes
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Transcript of AS Level Biology - 3) Enzymes
3. EnzymesAnd the Kinetic Affinity
What are Enzymes
Biological CatalystSpecific a certain substrate by its R groupGlobular protein – water solubleRemain unchanged after the reactionsEnzymes can break and bond!Nearly all metabolic reaction are enzymes-catalyzedEnzymes reduce activation energy – increase rate
constant
RATE = K[A]*[B}y
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.
Activation Energy
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
The Lock and key Theory
1. The enzyme has a cleft/ depression called the active site2. Active site and the specific substrate has complementary shape
3. The substrate meets the enzymes by random movement4. The substrates fits into the cleft
5. The R group binds with the substrate6. An enzyme-substrate complex is formed
7. The enzyme catalyzes the reaction – breaking it apart or joining8. An enzyme-product complex is formed
9. The product leaves the enzymes10. The enzymes remain unchanged – ready to go for the next substrate
Lock and key theory
The Induced Fit Theory
Like the Lock-and-keyOnly here, it is recognized the enzyme is
more flexible – able to change shape slightly to fit the substrate
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
Increase in temperature
Molecular movements speed up – more random movements – more activities
37 degrees – the optimum for bodily enzymesUntil up to 40 degree Celsius, all is good, rate
of reaction proportional to temp. – where the hydrogen bond breaks - denature
Decrease in temperature
Less active enzymesHowever – this do not denature the enzymesCertain animals can work with this
(Psychrophilic like cold, thermophiles like it hot, hyperthermophils can not grow anywhere lower than 70 degree Celsius)
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.
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
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
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.
Enzyme Kinetics
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
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
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
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
Steps to doing this
What we expect…Enzymes with higher substrate concentration
would work fasterWhen 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
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
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.
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
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.
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
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
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
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
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
Non - Competitive
Change the enzymes formationCan have both bounded at the same time
(Enzyme-Substrate-Inhibitor can form but the enzymes do not work)
No reduced affinity – Km stays the sameHowever since product can not be produced –
Vmax decreases
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
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.