Kinetic Considerations – Enzyme Catalysis

by Kevin Ahern, PhD

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    00:00 When we saw the image of the reaction occurring, we saw these various states that you see on the screen.

    00:04 The enzymes plus the substrate bounds together to make the ES complex, which converted upon the change in the enzyme to the ES* complex, which created the EP or the enzyme product complex, which ultimately resulted in the release of the enzyme and product. Now I comeback to this, because, we are going to need to consider somethings about the kinetic parameters that is the speed of parameters of the reactions that we are going to study.

    00:32 Now this rate of formation of product is really what we are interested in.

    00:37 When we talk about how fast an enzyme an make a reaction occur, this is the guts of what we are after. We wanna know how fast is the enzyme able to do this.

    00:44 Well, if to do this we need to make some simple assumptions. So we assume in the simple case that the enzyme substrate complex proceeds directly to enzyme plus product, okay? So when we simplify this more complicated equation above to a simpler equation below and this is done to help us better understand what's going on in the overall mechanism.

    01:07 Now these constants that are here won't really enter into our consideration.

    01:13 But they kcat that you see in the enzyme going to E plus P will, in fact, be an important consideration for us as we talk about the kinetic parameters.

    01:24 The kcat, as we shall see, is the rate with which product is forming.

    01:26 Now let's consider what's happening inside of a couple of different scenarios of a reaction.

    01:35 We can imagine that we have enzyme, for example shown in yellow. And we have substrates as little red color balls that are there.

    01:43 We can have a situation, first of all, where we have a reaction going on in the condition of low substrate.

    01:48 And if we have a low amount of substrate in a solution, we could imagine that there is very few enzymes that are going to be bound a substrate; because, the chances of encountering a substance are reduced.

    02:00 In the middle, of course, we have an intermediate state where we have a little bit higher concentration of substrate than we did before.

    02:05 And so we can see here that there are more enzyme molecules bound to and engaged in the process of making the product. And the third scenario we could imagine is high substrate.

    02:17 And we have a situation of high substrate. We notice here that every enzyme is bound to a substrate.

    02:27 And that's important because at high substrate concentrations we have enzymes that are what we call saturateable substrate.

    02:33 Meaning that once it is bound to substrate made a product and released it, almost instantaneously it grabs another substrate. It's not sitting around and waiting for things.

    02:46 Now, so enzymes interestingly have some kinetic considerations which is of course what we are interested in studying here.

    02:56 But we are seeing now for the first time a projection of the way that the enzyme is working. So I need to explain somethings on the graph that you see.

    03:03 First of all, we are plotting on this graph a reaction. The reaction is plotting the velocity of the reaction on the y-axis verses the substrate concentration that's used in the reaction on the x-axis.

    03:17 Now you notice the V has a little 0 beneath it. And the 0 beneath it, I will explain later. But it's called the initial velocity for our purposes. The velocity of a reaction is measured as the concentration of product made, divided by time. The concentration of product made per time. But we measure concentration in molar, mini molar, micro molar, etc.

    03:42 So that will be some morality per time. That's how the velocity is measured.

    03:47 The substrate concentration varies because, to generate a curve like this, I do not one reactions, but I do series of reactions.

    03:55 So let me set that up. We could imagine for example that I am setting up a series of 20 reactions, 20 different test tubes. I want to measure the velocity in each one of those test tubes.

    04:07 And what I do is I take into that test tubes, I place the buffer that holds the substrate.

    04:14 I place the substrate and I place the enzyme.

    04:18 Now when I am doing an experiment I wanna have one variable; because, one variable is the only thing I can really manipulate and measure the effect of that.

    04:29 The variable I have here is substrate concentration. I used the same amount of enzyme in every tube.

    04:34 All 20 tubes have the same amount of enzyme. They all had the same amount of buffer and they have varying amounts of substrates, starting from small amounts to very high amounts.

    04:46 I take and I let each one react for an exact same time and then I measured the amount of product.

    04:54 So by doing that I can see the effect of changing substrate on the velocity and then I plot it.

    05:02 So what you see on the screen is the sum of those plots, that is, each point on that dot came from a series of reactions that I did.

    05:09 And each one of those individual reactions have a specific substrate concentration and a specific velocity that was reached. Well not surprising as we look at this what do we see.

    05:21 Well, on the far left we are at low substrate concentration. What's the velocity? It's very low.

    05:25 And that's what I showed on the original image.

    05:29 Low substrate concentration, enzyme is sitting there waiting for substrate there is not gonna be much velocity.

    05:37 When I get to a high substrate concentration such as I see on the right side of the screen I have got a high velocity. Make sense. Okay, low substrate, low velocity; high substrate, high velocity. I want you to remember that.

    05:49 Now I am showing another plot here to illustrate a principle of a reaction.

    05:54 On the y-axis, I have the concentration of product, we can think of that again as the velocity.

    05:59 But on the x-axis now, I am plotting the time of a reaction.

    06:03 So I can take one of the tubes that I used in the previous one. And for example, look at how fast the product is being accumulated and what happens with that product over time? We can see on this plot that over the early range of the reaction, there is a linear relationship between the production of product and time, okay? But after a while what happen is that curve flattens on.

    06:29 Now what that means is that the longer that we let a reaction go it doesn't stay linear forever. And the reason it doesn't stay linear forever because remember enzyme catalyze reversible reactions. So the more we let product accumulate the more likely product will start being converted back into substrate.

    06:50 Well, that's not what we are interested in studying. We wanna study how fast the enzymes makes product.

    06:56 So for going to study an enzymatic reaction we have to study what's called initial velocity.

    07:03 We don't wanna wait too long in order to study the concentration of product, because if we wait too long, we are actually starting to study the reverse reaction and that's not what we are after.

    07:13 So that's why we used Vo or the initial velocity in our measurements. Okay.

    About the Lecture

    The lecture Kinetic Considerations – Enzyme Catalysis by Kevin Ahern, PhD is from the course Enzymes and Enzyme Kinetics.

    Included Quiz Questions

    1. Aspartic acid and histidine play important roles
    2. The alkoxide ion forms on aspartic acid
    3. Electrophilic attack is at the core of the reaction mechanism
    4. The slow step is the first one
    1. It determines the binding/cutting specificity of the enzyme
    2. It stabilizes an unstable tetrahedral intermediate
    3. It is the location of the catalytic triad
    4. It requires serine for binding
    1. The rate of formation of the product
    2. The rate of formation of the enzyme-substrate complex
    3. The rate of formation of the enzyme-product complex
    4. The rate of disappearance of the product from the reaction mixture
    5. The rate of backward reaction
    1. The product starts being converted back to the substrate
    2. The substrate concentration reaches to zero with the passage of time
    3. The enzyme gets tired of working over and over with the substrate molecules
    4. The enzyme undergoes major conformational changes, so it becomes unable to bind the substrate anymore
    5. The enzyme starts to degrade

    Author of lecture Kinetic Considerations – Enzyme Catalysis

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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