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.