Michaelis-Menten Kinetics: Parameters – Enzyme Classification

by Kevin Ahern, PhD

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    00:01 Okay well let's now look at these parameters. I have introduced the concept of Vmax and we see that eventually the enzyme reaches a place where it's not gonna make any more product over time; because, it's saturated with substrate.

    00:14 Vmax turns out to be an interesting quantity but Vmax as we will see has some limitations.

    00:20 Nonetheless, Vmax allows us to study some things.

    00:24 Now the quantity Vmax gives us a maximum amount and we can say "Well, if we wanna understand how much an enzyme interacts with a substrate maybe we should compare Vmaxes." Well that doesn't really tell us very much. It tells us how fast a reaction goes.

    00:40 But it doesn't tell us how well an enzyme interacts with a substrate; because, any enzyme will reach Vmax as we add an infinite amount of substrate which is theoretically what Vmax is occurring at, when it is completely saturated.

    00:56 That doesn't tell us very much. However the quantity Vmax/2 where we are getting an enzyme to a certain point of velocity, but not the maximum amount of velocity, actually allows us to measure that the affinity that enzyme has for its substrate.

    01:11 If we compare a variety of enzymes and we compare how much substrate it requires the enzyme to get to Vmax/2, we get something very interesting. We get a quantity called the Km.

    01:24 And the Km is actually a measure of the enzyme's affinity for its substrate. So I say affinity is the desire to bind to.

    01:33 How well does it bind to its substrate? Now Km is interesting. If we think about two enzymes one enzyme that catalyzes a reaction that has great affinity for its substrate. It really likes that substrate. It really grabs that substrate.

    01:48 And we have another enzyme over here that doesn't like its substrate as well, okay? Well which of the two are gonna bind substance more readily? The first one, of course; because, it has got greater affinity.

    01:59 Which one is going to get to Vmax/2 with a lower substrate concentration? Well the one that grabs its substrate more easily.

    02:07 So enzyme that have a greater affinity for their substrate are going to have a low Km.

    02:13 And those that have less affinity for their substrate are going to have a higher Km.

    02:19 Okay? Greater affinity, low Km; lower affinity, high Km. So Km is inversely proportional to the enzyme's affinity for its substrate.

    02:32 Okay? So here we see high Km, low affinity.

    02:36 We see low Km, high affinity.

    02:38 A very important concept to remember with respect to Km.

    02:42 And I would like to think about an enzyme that has low affinity we have to pounder on the head with substrate before it starts to bind it.

    02:50 And by poundering on to head, the way we do that is by adding a lot more substrate.

    02:56 Now Vmax, as I said, is a very interesting and important quantity.

    03:00 But it actually is not the perfect quantity to measure the speed of a reaction. It's good for the reaction but it's not so good for the enzyme.

    03:09 So what does that mean? Well it means that Vmax when we a do a reaction, the way I described doing a reaction, is we set up 20 tubes, and we have in those 20 tubes, buffer, we have substrate, and we have enzyme.

    03:25 And when we are doing a V versus S plot, what we are doing is we are having one variable.

    03:29 The one variable that we have is substrate, which means that all 20 tubes have the same amount of enzyme.

    03:37 That's great. We don't wanna have variable amounts of enzyme.

    03:41 But imagine I would do the same set of reactions.

    03:44 And instead of using the amount I used in the first set Let's say that I did the reaction and I used twice the amount of enzyme for the second set of reactions, in each case constant, however, varying substrate but now with twice the amount of enzyme.

    03:59 What would I see with respect to Vmax? Well if I go back to my factory analogy, and I think about what happen with the factory, I said that the factory got to a point where it's saturated. It made a maximum amount of product that the workers are gonna put out per day and it wasn't gonna make any more.

    04:15 What if I had two factories? Well if I had 2 factories I would say "Well I'd probably expect that I will get twice as much product per day".

    04:23 And so I if used a set of tubes that have twice as much enzyme, the parallel follows. I would get twice as much product.

    04:31 So Vmax is proportional to the amount of enzyme I use. It's not a constant for an enzyme.

    04:36 But it's a constant only for a reaction with a set amount of enzyme.

    04:40 I would like to be able to compare enzymes with a quantity that is independent of the amount of enzyme that I used.

    04:46 Well fortunately that's fairly easy to do.

    04:49 Okay? Vmax is a velocity and we measure velocity of a reaction as the concentration of the product produced divided by time.

    05:00 If I take the quantity of enzyme that I used in the reaction and I divide Vmax by that quantity, I said quantity in this case meaning concentration, the concentration of enzyme that I used, what will happen? Well, the Vmax was measured as a concentration of product and I divided by a concentration of enzyme, as I long as I used concentration and concentration consistently the concentrations actually drop out.

    05:25 And so what happens is, I get a number and the units on the number are per time.

    05:32 So I get something it says a 1000/second.

    05:36 What does a 1000/second mean? Well, I have taken the enzyme out of the equation and now the number that I get corresponds to the number of molecules of product per enzyme per second.

    05:50 So a 1000/second means every enzyme in that solution is making a 1000 molecules of product per second.

    05:58 And that's the fastest it's gonna go, because, remember we started with Vmax.

    06:02 That quantity is called Kcat.

    06:05 Kcat is a number that's also called the turnover number.

    06:10 But I can compare the Kacts of two enzymes and have a much better understanding about the relative speeds of production of product that those enzymes have.

    About the Lecture

    The lecture Michaelis-Menten Kinetics: Parameters – Enzyme Classification by Kevin Ahern, PhD is from the course Enzymes and Enzyme Kinetics.

    Included Quiz Questions

    1. It has no relation to the substrate concentration
    2. It is not proportional to the affinity of the enzyme for substrate
    3. It varies with substrate concentration
    4. It increases as the amount of enzyme decreases
    1. They give hyperbolic plots of velocity of a reaction vs subtrate
    2. They are ones where the substrate changes the enzyme’s binding affinity when it is bound
    3. They have varying Km values
    4. The have constant Vmax values
    1. An enzyme’s affinity for its substrate
    2. The amount of enzyme required to get to Vmax/2
    3. The substrate necessary for Vmax
    4. The amount of enzyme necessary to get to Kcat
    1. Kcat is independent of the amount of enzyme used in a reaction
    2. Vmax requires calculation of Km/2
    3. Kcat is directly related to the amount of enzyme used.
    4. Kcat/2 gives a more accurate measure of Km

    Author of lecture Michaelis-Menten Kinetics: Parameters – Enzyme Classification

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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    By Hazazi M. on 04. October 2018 for Michaelis-Menten Kinetics: Parameters – Enzyme Classification

    THANK YOU Pro Ahern you're amazing! very well explained!