Perfect Enzyme and Lineweaver Burk Plot – Enzyme Classification

by Kevin Ahern, PhD

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    00:01 Now the idea of Kcat brings up another thing for us to think about and enzymes are really remarkable. Okay? We have seen that enzymes can speed up reactions mind-boggling numbers of times.

    00:13 And we have also introduced the concept here of an enzyme having affinity for its substrate.

    00:21 The idea of what a perfect enzyme would mean, starts to come into shape. We think about "What would be a perfect enzyme"? A perfect enzyme would be an enzyme that would have as much velocity as possible with as great an affinity for its substrate as possible. Meaning that to get them maximum velocity it wouldn't take very much substrate; because, the enzyme would be grabbing substrate and converting it into product very readily.

    00:48 So a perfect enzyme would have a high velocity and a low Km.

    00:54 Well we use Kcat as our measure of velocity and Km is our measure of affinity for substrate.

    01:02 High Kcat means high velocity, low Km means high affinity.

    01:10 The perfect enzyme will have a large ratio of Kcat to Km.

    01:15 So if we take those two numbers and we divide them by each other, and we start by comparing enzymes, we see enzymes have widely varying ratios of Kcat/Km.

    01:26 But we also see that there is a sort of top echelon beyond which enzymes really don't have a number that increases very much. Now these numbers are very a little bit from each other. But these are really the top echelon enzymes.

    01:40 They don't have a Kcat/Km value that's significantly different These running orders of 10th to the 7th, in one case 10th to the 9th, but most in the range of about 10th of the 8th.

    01:49 We don't see enzymes are making to the attempt to 15th for example.

    01:53 Why is that? Well, what's happened with these enzymes is they have reached their maximum efficiency.

    02:02 They can't get any more efficient. There are two things that limit them.

    02:06 One is they can't with shape and sequence of amino acids, make a better active site then what they have made by evolution.

    02:14 In that sense they literally are perfect.

    02:17 Mutations that change those will always make an enzyme that's less efficient.

    02:23 There is a limit to what that efficiency can be. The second thing is really interesting.

    02:28 It is believed that the reason that we reach a max with this in addition to what I have just mentioned is that there is something else that is limiting about the enzymatic reaction.

    02:38 And the limiting thing for these enzymes in a solution is 1 quantity.

    02:45 And that's the rate with which the substrate can diffuse in water.

    02:49 Diffusion of course happens with the mixing that we see.

    02:52 In this diffusion that's bringing substrate into the enzyme's active site.

    02:56 And though that process of diffusion can itself occur at mind- boggling rates, that's what allows enzymes to do what they do, it, too, has a limit.

    03:04 And so these enzymes are so efficient that they are sitting, they're waiting on water to deliver substrate to them. That's a remarkable thing.

    03:13 Alright let's take and use now of some of these parameters that we have been talking about with respect to kinetics and understand enzymatic reactions.

    03:22 I have shown several times now the plot of Vo versus S and we saw that was a hyperbolic curve.

    03:27 And you saw in that curve that at the very top of that, we had something called Vmax.

    03:33 And if I am eyeballing that curve I have to ask myself "Well, have I drawn Vmax at the right place? Is it up a little bit? Is it down a little bit?" and I have to make a judgement call with that.

    03:43 I would like to have a more precise way of saying "What is the Vmax?" Well, one of the tricks or tools that we used to do this, is to actually change the analysis of the data a little bit.

    03:53 Instead of plotting Vo versus the concentration of substrate, that is the velocity versus the concentration of substrates, I take the same data that I had for that Vo versus S plot.

    04:06 And I invert it. I invert all the datas. So I do what's called a double reciprocal plot or a Lineweaver Burk Plot. They were the people who came up with this.

    04:16 And when I invert the data like that, what I discover is that the hyperbolic plot becomes linear.

    04:22 Now a linear plot is much more easy for us to interpret to determine what these values are.

    04:28 When I make such a double reciprocal plot, I create a linear plot of the data and the linear plot of the data I can draw a line through the points and extrapolate through the axes, the y-axis and the x-axis.

    04:45 When I do, I create an intercept on a y-axis and the y-axis has the value of 1/Vmax.

    04:52 I can very quickly, of course, invert that value and I have got Vmax.

    04:56 On the x-axis the intercept is -1/Km.

    05:01 So if I take whatever that value the intercept is and I take -1 over that I would get Km. Very simple plot. So Lineweaver Burk Plots. And there are other manipulations that people do a graphs.

    05:11 Lineweaver Burk Plots help me to very readily determine Vmax and Km from a set of data.

    About the Lecture

    The lecture Perfect Enzyme and Lineweaver Burk Plot – Enzyme Classification by Kevin Ahern, PhD is from the course Enzymes and Enzyme Kinetics.

    Included Quiz Questions

    1. It has a high Kcat and a low Km
    2. It has a high Vmax and Km
    3. It has a low Kcat and a high Km
    4. It is limited only by the diffusion of the enzyme in the solution
    1. The Km is the negative reciprocal of the value of the X-intercept
    2. Vmax is equal to the value where the line crosses the Y-axis
    3. The slope of the line is equal to Kcat/Km
    4. Kcat is equal to the value of the X-intercept

    Author of lecture Perfect Enzyme and Lineweaver Burk Plot – Enzyme Classification

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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    Very Clear Explanation!
    By Ilay B. on 13. March 2019 for Perfect Enzyme and Lineweaver Burk Plot – Enzyme Classification

    I finally understood why we have Lineweaver Burk Plot and how we read it.