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Enzyme Kinetics
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0:00
Hi, this is Dr. Vijay. In this video, we're going to study about an important topic in enzymology, that is enzyme kinetics. What is this enzyme kinetics? So, enzyme kinetics is study of enzyme catalyzed reaction rate, essentially, we are studying the rate of reaction in varying condition, in different conditions with the experimental parameters. Study of enzyme catalyzed reaction rate in varying conditions. The varying condition means that changing experimental para meters, for example, so
0:55
if you change the pH, what will happen to the rate of enzyme catalyzed reaction. So, this study is nothing but enzyme kinetics. Similarly, we have temperature, all these experimental para meters there is going to be change in the velocity or rate of enzyme catalyzed reaction, even concentration of substrate OK whenever I use substrate concentration, I will use this abbreviation [S], enzyme concentration
1:30
also, so we use this [E] for enzyme concentrations, like there are different experimental para meters, even the product concentration also change the rate of enzyme catalyzed reactions. We will study enzyme kinetics today by taking the substrate concentration with the enzyme concentration being constant. We will understand certain aspect of enzyme kinetics, there is a constant called Michaelis-Menten constant and there is an equation.
2:08
All this aspect we're going to study with taking one experimental condition, that is varying [S] concentration. We will study the enzyme catalyzed reaction rate with different substrate concentrations, provided enzyme concentration is constant. Here I already draw the graph. So, in the X axis we have different substrate concentration. Similarly, in the Y axis we have reaction rate or velocity and the initial velocity, we can write it as V0 also can write it as Vi.
2:58
So, you may find Vi or V0 for initial velocity in different textbooks, both are same. So, that means we have plotted here the rate of enzyme catalyzed reaction with varying substrate concentration and you need to remember here the enzyme concentration is constant. So, after plotting the rate of reaction against varying substrate concentration, we got a curve, which is called hyperbolic curve. You can see this curve, that means the rate or velocity of reaction assumes hyperbolic curve.
3:51
Initially the increase in the velocity or rate of reaction is almost linear or directly proportional but as the substrate concentration increases the rate of reaction assumes a steady state, there is a plateau formation here. You can see it is, at a relatively low concentration of substrate the velocity increases almost linearly with the an increasing the substrates. Remember enzyme concentration is constant here.
4:31
So, this linear increase or directly proportional increase of rate of enzyme catalyzed reaction is called first order kinetics. So, why there is a linear increase of rate of enzyme catalyzed reaction with increasing the substrate initially because almost all enzymes active sites are empty, so they can occupy with the substrate. So, there is increase in the rate of reaction at relatively low concentration of substrates,
5:12
this is called first order kinetics but at higher substrate concentration, you can see if you add more and more substrate with a constant enzyme concentration, the increase in the rate of reaction is not linear it assumes a hyperbolic curve and finally there is a plateau formation. So that means the higher the substrate concentration, increase in the velocity is less and less with the increase in the substrate.
5:52
Finally, a point is reached beyond which increasing the substrate concentration cannot make any effect on the velocity. You can see even if you add more and more substrate here the velocity or rate of reaction is constant, it assumes a steady state. So, this is called zero order kinetics. So, this is a steady state now and this point where the plateau formation or the zero order kinetics, so this point we can call it as Vmax OK, if you connect this plateau or steady
6:41
state to the Y axis, a point is called maximum velocity or we can abbreviate it as Vmax. So, initial velocity is Vi or V0, as the substrate concentration is increased, there is increase in the rate of enzyme catalyzed reaction or increasing the velocity. Initially almost it is a linear but as you increase substrate concentration, increase in the velocity going to be less and less and less and finally it assumes maximum velocity or zero order kinetics. Why this happens?
7:26
Why it assumes steady state? Imagine initially, we already written here enzyme concentration is constant but initially when less substrates are there, there are more enzymes or more active sites to the substrates. When concentration of substrate increases but enzymes concentration is constant, in the steady state, virtually all enzymes are in enzyme substrate complex. So, there is no free enzymes or there are no free active sites for the substrates.
8:12
So, that is why you can say there is no increase in the rate of reaction. In other words, we can say enzymes are saturated. You may say this enzyme substrate complex broken down into enzyme and products, even that enzyme is free, immediately this substrate can bind to this free enzyme. So, you can say in steady state or zero order kinetics almost all enzymes are saturated, they are in the form of enzyme substrate complex.
8:52
Initially there is increasing the rate of reaction linearly because there are many free enzymes available for the substrates. So, this curve is a hyperbolic curve. So what is the use of this plot? So, with the help of effect of substrate concentration or effect of varying amount of substrate concentration with constant enzyme concentration, we can derive some of the terminology and it can be used in experimental condition, especially in the vitro or in laboratory.
9:32
So, if you take half of this maximum velocity in the Y axis and connect this point to the graph and if you connect this point from the graph vertically down to the X axis, we will reach a point and take point we can describe it as Km. So, what is Km? At this point Km is half the Vmax, from the Y axis we have taken midpoint of maximum velocity and we connect this point to the hyperbolic curve and traced down vertically to the X axis. So, X axis we know that it is a substrate concentration.
10:20
So, this Km is a constant, this constant is also called Michaelis-Menten constant. This Km is very very important, that means it is a substrate concentration at which the reaction rate is half of its maximum velocity. So, Km is the substrate concentration at which the reaction rate is half of its maximum velocity or Vmax and there is a equation where we can calculate either Vmax or Km or the initial velocity, so that is called Michaelis- Menten equation.
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Initial velocity is equal to maximum velocity into substrate concentration divided by Km plus substrate concentration. So, you can remember this equation, you remember, very much sweet kills subject, in this way we can remember Vmax, very much, S for substrate concentration, K for Km and again S for substrate concentration. So, this is a Michalis-Menten equation but from this plot for us the important topic is Km or Michaelis-Menten constant. This Michaelis-Menten constant is called Km.
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So, what is Michaelis-Menten constant? Substrate concentration at which the reaction rate is half of its maximum velocity, half of its maximum velocity. So, maximum velocity is actually Vmax. So, this is the definition of Km, substrate concentration at which the reaction rate is half of its maximum velocity. What is the significance of this Km? It is a very important parameter in enzyme kinetics. Every enzymes have their own Km.
12:54
So that means Km is different for different enzymes not only different for different enzymes even same enzyme has got different Km for different substrates. Same enzyme has different Km for different substrates. The major significance of this Km, it provides the measure of substrate required for significant catalysis. It provides amount of substrate required for the significant catalysis to occur or enzyme
13:40
catalysis to occur and another most important significance of this Km it represents affinity of the enzyme for substrates. Affinity of enzyme for the substrate. So, what is the meaning of this affinity? So, let me explain here. I will take a enzyme hexokinase the substrate is here glucose. So, this glucose can be converted to glucose-6-phosphate either by hexokinase or by glucokinase in liver. So, these 2 enzymes, they have same substrate, that is glucose.
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If you take Km value of hexokinase, it is 0.05 mmol/L/ and the glucokinase Km is very high that is 10 millimoles per liter. What is the meaning of this? This is Km value. In hexokinase the Km value is very less whereas compared to hexokinase glucokinase has got higher Km value. So, whenever there is less Km value that means the 1st order kinetics occurs very very fast, that means the affinity of the substrate for the enzyme is very very high or the binding
15:21
of the substrate to the enzyme is strong. So, whenever there is less Km that means more affinity. Whenever, there is more Km that means less affinity or weak binding to the substrate. You should know all enzymes have their own different Km value for different substrate, even same enzyme can have different Km value for different substrates. Glucose can bind to hexokinase very easily and this binding is a very strong binding
16:06
and there is the increase in the rate of reaction to give glucose-6-phosphate, when compared to glucokinase. Glucokinase has weak or less affinity to the glucose when compared to hexokinase. So, let me explain this concept by taking a clinical example. So, I will take an example, if you see some of the alcoholics, they develop alcohol sensitivity. So, that means even if they take a very small amount of alcohol, they will end up with facial
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flushing, that is redness around the cheek and also there is increasing in the heart rate that is called tachycardia. Usually alcohol is metabolized in the liver. Initially alcohol converted to acetaldehyde by an enzyme called alcohol dehydrogenase and this acetaldehyde converted to acetate. So, this is the normal alcohol metabolic pathway in the liver. Now the conversion of acetaldehyde to acetate requires an enzyme called acetaldehyde dehydrogenase.
17:37
So, normally acetaldehyde converted to acetate in liver by acetaldehyde dehydrogenase. So, this alcohol sensitivity developed by accumulation of acetaldehyde in the blood that mean there is some problem in this enzyme. There are 2 forms of this enzyme that is one form is mitochondrial acetaldehyde dehydrogenase another one is cytoplasmic or cytosolic acetaldehyde dehydrogenase. So, that means there are 2 enzymes, we can call it as isoenzymes, one form of enzyme
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usually expressed from the liver mitochondria, and another one is expressed from the cytoplasm. The Km value of mitochondrial acetaldehyde dehydrogenase is very less, whereas the Km value of cytosolic acetaldehyde dehydrogenase is more. So, which has got more affinity for acetaldehyde? definitely mitochondrial acetaldehyde dehydrogenase has got more affinity for acetaldehyde, so that acetaldehyde will be converted quickly to acetate, that means there is no accumulation of acetaldehyde.
19:06
So, this alcoholic sensitivity subject there is a problem with this mitochondrial acetaldehyde dehydrogenase, so that means whatever acetaldehyde is formed from the alcohol in the liver it should be converted to acetate pipe cytosolic acetaldehyde Big head is formed from the alcohol in the liver it should be converted to acetate by cytosolic acetaldehyde dehydrogenase. We know that cytosolic acetaldehyde dehydrogenase has got more Km value that means here it is a less affinity.
19:41
The binding of acetaldehyde to the cytoplasmic acetaldehyde dehydrogenase is very very weak, so then there is accumulation of acetaldehyde in the liver, then in the blood. So, the increased concentration of acetaldehyde in the blood usually develops facial flushing and tachycardia even after taking very small amount of alcohol in these subjects or individuals. So, this is a significance or clinical significance of Km. so, enzyme kinetics is nothing but
20:18
study of enzyme catalyzed reaction rate or velocity in different condition or changing experimental parameters like pH, temperature, substrate concentration, enzyme concentration, product concentration, activators, inhibitors so and so. So, today I have explained by taking varying concentration of substrate with a constant enzyme concentration. We have learned what is Kim value and what is its significance. Thanks for watching.
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