AS Level Biology - 3) Enzymes

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

2. 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

3. 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.

4. Activation Energy

5. 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

6. The Lock and key Theory The enzyme has a cleft/ depression called the active site Active site and the specific substrate has complementary shape The substrate meets the enzymes by random movement The substrates fits into the cleft The R group binds with the substrate An enzyme-substrate complex is formed The enzyme catalyzes the reaction – breaking it apart or joining An enzyme-product complex is formed The product leaves the enzymes The enzymes remain unchanged – ready to go for the next substrate

7. Lock and key theory

8. 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

9. 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

10. 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

11. 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)

12. 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.

13. 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

14. 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

15. 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 .

16. Enzyme Kinetics

17. 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 V max INITIAL RATE OF REACTION IS THE THEORETICAL VELOCITY OF A REACTING ENZYME FOR EACH CONDITION

18. 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

19. 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

20. 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

21. 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

22. 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

23. Michaelis Menten Model When the Enzymes are working at the hardest, and they can not go any faster - E nzyme saturation This is the Vmax – a maximum rate in which an enzyme can work at.

24. V max 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

25. K m Vmax/2 is K m K m measures the affinity/ efficiency of an enzyme – how quickly an enzyme reaches V max It only points to when a substrate is already in an enzyme Kinda like acceleration – how quickly it reaches the maximum speed.

26. 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 V max - 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/V max . -1/K m (because we can’t have negative K m ) can be found at the point of x-axis interception

27. 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 V max (a theoretical maximum velocity of a certain enzyme) and how quickly the enzyme can reach that, expressed in the terms of K m K m = ½ of V max – calculated by the reciprocal plot or a hyperbolic normal plot

28. 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

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 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

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

31. 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 – K m stays the same However since product can not be produced – V max 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 eg. When maltose inhibits amylase

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