Lesson video

This lesson is called "The Effect of pH On The Rate Of An Enzyme Reaction: Data Analysis," and is from the unit Biological Molecules And Enzymes.

Hi there.

My name is Mrs. McCready, and I'm going to guide you through today's lesson.

So thank you very much for joining me.

In our lesson today, we're going to analyse and explain data that we've collected from an investigation into the effect of changing pH on the rate of enzyme reaction.

We're going to cover a number of key words in our lesson today.

Now, they're presented on the screen.

You might wish to pause the video to make a note of them, but I will introduce them to you as we go.

Now in our lesson today, we're going to first of all interpret the experiment data and then we're going to explain the effect of pH on enzyme reaction.

So I hope you're ready to go, I am.

Let's get started.

So we know that enzymes are biological catalysts.

That means that they speed up the rate of a chemical reaction turning substrates into products, and we can state that the rate of a reaction is the measure of how much change occurs per unit of time.

So how much substrate is changed into product.

So how much substrate reduces or how much product is increasing within our sample, for instance, that would be a measure of the rate of the reaction.

So in the reaction catalysed by the enzyme amylase, we can see that starch is converted into maltose, which is a type of sugar.

So a long starch molecule is converted into maltose by the enzyme amylase, and we can predict that the rate of this reaction will change at different pHs.

So which statement defines the term rate of reaction? Is it A, how long a chemical reaction takes? Is it B, how much substrate is converted into product, or is it C, how fast a chemical reaction occurs? I'll give you five seconds to consider this.

Okay, so hopefully you have decided that the term rate of reaction is described by statement C, how fast a chemical reaction occurs.

Well done.

So in this experiment where we're taking starch and we're subjecting it to amylase, the enzyme, which is going to convert it into maltose, we can change the pH at which is this reaction is taking place, and then make a prediction about what effect that pH will have on the rate of the reaction.

So we can predict that at the optimum pH rate should be highest, and at other pHs the rate will be lower, will be less.

And so we're going to analyse some data from this experiment and decide whether it either increases or decreases our confidence in this prediction that at the optimum pH, that is where the rate will be highest.

And at the other pHs, the rate of reaction will be less.

So in this experiment, we continuously sampled the reaction mixture.

So what does that mean? So what we did was take a sample of the reaction mixture every minute and added it to a spotting tile with 10 wells filled with iodine solution.

Now, iodine turns blue black, in the presence of starch and remains brown orange, when no starch can be detected and in the reaction starch is being broken down into maltose.

So the point at which all of the starch has been broken down into maltose is called the end point.

Because the end of the reaction has been reached, there is no more starch to break down and therefore the reaction has finished.

Now at this point, because there is no starch present, it's all been digested down to maltose that we would expect to see no colour change happening within the iodine solution because there's no starch to make the iodine change colour.

Remember that iodine changes from brown orange to blue black in the presence of starch and therefore won't change colour if there is no starch present.

So we can see at the very beginning of this experiment, iodine is turning blue black because starch is present, but as the reaction continues, there's no starch remaining and therefore iodine does not change colour.

And we can see that by about nine minutes and certainly by 10 the iodine is remaining that orange brown colour.

So let's see some experiment results then.

We can see that at pH seven, the time it took for all of the starch present to be digested into maltose by the enzyme amylase was three minutes.

The endpoint was reached by three minutes.

It probably happened earlier, but we are only testing once every minute.

So we can't say any more definitively than that.

But at pHs two and 12, so really quite extreme pHs, the iodine simply continued to change colour to blue black, and so the endpoint was never reached.

So we can note down that therefore the endpoint is more than 10 minutes.

We can't say if it's 11 or 12 minutes or if it never happened at all because we've stopped testing at 10 minutes.

But we can say that it's certainly more than 10 minutes as we can see in the table there.

So we can plot that data on a graph.

So we're gonna put pH our independent variable, the variable that we've changed on the X axis as usual, so pH on the X axis, and then our dependent variable.

So the measurement that we're taking on the Y axis in this case, it's the time taken to reach the endpoint in minutes.

Then we can plot these points onto the graph.

So at pH seven we can plot that it took three minutes for the reaction to complete, for the endpoint to be reached, whereas at pH two, it took more than 10 minutes.

And at pH 12 it also took more than.

So I'm just going to put those points at the 10 minute mark because I can't say for sure how many minutes it actually took took because we're just going to put them at 10 for now.

Then we can plot a line of best fit through those points and create this kind of U shape on the graph.

Now you don't tend to see graphs of that shape when they're describing enzyme reaction rate, and that's because what we were showing was the time it took for the reaction to take place rather than the rate itself.

So it's possible to convert the time taken into rate, and that's what scientists generally do.

Now, to do that, we need to divide one by the time taken.

So one divided by the time taken will give us the rate of the reaction, a very straightforward calculation, but it can result in extremely small numbers.

So we can do this with the data from our experiment.

So the at pH seven, the time taken was three minutes.

So if we take one and divide it by three to one decimal place, that will give us a rate of reaction of 0.

3.

Now for pHs two and 12, the time taken was more than 10 minutes.

So we don't actually have a value that we can divide one by.

And for this reason, I'm going to say that the rate of reaction was zero, that in other words, either no took place or it took place so slowly that we haven't been able to measure the rate of the reaction.

So I'm just going to put zero down for the rate of those reactions at pHs two and 12.

We can now plot the rate of reaction values on a graph instead of the time taken.

So let's see what that would look like.

So again, we're putting our independent variable of the pH values on the X axis as usual, and this time we're going to plot the rate of reaction up upon the Y axis.

So this is a value between zero and one.

Then at pH seven, we can say that the rate of the reaction was 0.

3 and mark that on our graph.

And at pHs two and 12, there was zero rate of reaction.

So we can also plot those points, then add a line of best fit to curve neatly between those three points.

And that looks much more like a reaction rate graph that we would be used to seeing within textbooks and exam papers and various other published items as well.

So our original prediction was at the optimum pH, the rate should be highest, and at other pHs the rate will be less.

Now if we take our results as plotted on the graph, we can take our prediction and we can compare it to those results.

So let's just do that.

At the optimum pH, the rate should be highest.

Well, we can say that as far as we are able to tell that is true because the highest rate was about pH seven.

And so we're going to assume that the optimum pHs in the vicinity of pH seven and at other pHs, the rate will be less.

Well, we can say that for sure because we can see at pHs two and 12 the rate is zero compared to 0.

3 at the notional optimum pH.

So we can say therefore that our results increase our confidence in our original prediction because our results confirm our predictions on both points.

But could we improve our confidence any further? And if so, how? So if we did want to improve our confidence in our results further and therefore our confidence in our prediction, we could repeat the experiment and calculate a mean of the rate.

We could test using a far greater range of pHs.

So instead of just testing two, seven, and 12, we could test one, two, three, four, five, six, seven, eight, nine, 10, 11, and 12 for instance, or even maybe six and a half, seven and seven and a half to get a more precise measurement of what the optimum pH might be.

So there's lots of variations on how we would actually do that, but we could test certainly using more pHs.

We could also sample more frequently.

So we were sampling every minute, but we could sample every 10 seconds for instance.

And then we get a much more specific idea of when the reaction actually started to reach its endpoint because instead of just having a measurement based on minutes, we'd have it based on seconds instead, or we could not bother with the manual version and we could use some technology instead such as a data logger to do all the recording of the measurements for us and essentially get continuous data every second.

We would have a value taken using the data logger and that would give us much more data for improving our confidence.

So what I want you to have a look at now are these two graphs.

So which one is drawn correctly and of the one which you think is drawn incorrectly, what is it that is incorrect about that graph? So I'm gonna give you a few moments to study those graphs carefully.

Do take your time, pause the video if you need to.

Okay, so hopefully you decided that the correct graph is graph B, but why is the other graph incorrect? So hopefully you've identified that the axis, first of all, around the wrong way, the dependent variable has been put on the X axis where it should always be put on the Y, and the independent variable has been put on the Y axis when it should always be put on the x axis.

In other words, the rate of reaction and the pH scales need to be on the other axes.

Secondly, you should have noted that the scale for the rate of reaction is incorrect.

This is going up to a value of 10, but actually the rate of reaction will go up to a value of one and therefore have decimal places with it.

Well done if you spotted all of those errors and were able to correct them as well.

Good work.

So what I'd like you to do now is to use your data or the data provided and calculate the rate of the reaction using the equation rate equals one divided by time taken.

Then I'd like you to plot a graph of the rate against pH and draw a smooth line of best fit.

Now, your success factors for your graph are that the axes are correctly plotted and labelled that the points are plotted correctly and the smooth line of best fit is drawn on the graph too.

Then I'd like to consider either the prediction that I'm giving you or the one that you made and decide whether your results increase or decrease your confidence.

Then I'd like you to explain why you have come to that conclusion and how we could improve our confidence further.

So if you are using my prediction, that is that at the optimum pH, the rate should be highest and at other pHs the rate will be less.

So pause the video, take your time, be really careful with your calculations, and also when plotting your graph, remember that if you didn't get any change of results in more than 10 minutes, then use rate as zero and when you are ready, come back to me.

Okay, so let's see what we've done with this.

So if you are using your data or data that's been provided to you, you should have calculated rate using one over time taken and your results should look roughly like this, where if the time taken was three minutes, then the rate of the reaction is 0.

3.

And remember that any reactions that never actually finished as far as you are able to record, so where the iodine was always turning blue black after 10 minutes, then record those rates as zero.

Then I asked you to plot your rate on a graph against pH and draw a smooth line of best fit.

So your graph should look something like this with pH on the X axis rate of reaction on the Y up to maximum value of one, and then your points plotted and a smooth line of best fit drawn.

So remember, your axes need to have been correctly plotted and labelled.

The points need to have been plotted accurately and a smooth line of best fit drawn.

So check your graph against those criteria.

And then finally, I asked you to consider the prediction and decide whether your results increase or decrease your confidence, and how might you have improved that confidence further.

So you might have included that your results support your prediction or maybe you had to say that they disagreed with your prediction.

Maybe that's depending on what your prediction was and therefore that they either increase or decrease your confidence in your prediction that the rate is fastest at the optimum pH.

And as pH moves away from the optimum, the rate decreases.

And if we wanted to increase our confidence further, we could repeat the experiments using other pHs or we could repeat each experiment several times and then calculate a mean, both of which would increase our confidence further.

So review your answer against those criteria and well done.

That was quite a complex task.

Okay, let's move on to explaining the effect of pH on enzyme activity.

So we've seen that enzymes are catalysts, they speed up the rate of reaction and we should know that enzymes are proteins as well, and therefore they are formed from a very long chain of amino acids which are joined end-to-end, and then they're folded.

That chain is folded into a very specific shape.

So in the picture there on the screen, you can see the overall enzyme.

And then if we zoom in, you can see that long chain of amino acids joined end to end.

And if we zoom in a bit more, we can see that there are bonds formed between non-adjacent amino acids.

So these are additional bonds that are holding the protein in shape beyond the ones that adjoining one amino acid to the next.

So there are these extra bonds, and it's these extra bonds which are maintaining the shape of the protein, keeping it in the right shape that it needs to be in order to catalyse the reaction that it's supposed to be controlling.

Now, maintaining that shape is really important, and that's because part of the enzyme shape is called an active site and it's at the active site where the chemical reaction actually takes place.

So the substrate needs to fit into that active site.

And then the products are released once the chemical reaction has been catalysed.

Now that active site is a very particular shape.

It's incredibly precise, and it means that only one substrate will fit into that active site.

So just as this is just like one key will only fit a particular specific lock, we've got that same idea happening between the substrate and the enzyme.

And you can see there in the diagram lots of other different substrates which will fit into other enzymes, but they will not fit into this enzyme because only one substrate will fit into the active site of this enzyme.

Now that shape, as we've seen, is maintained by the bonds between non-adjacent that's not next to amino acids, and those bonds that are maintaining that shape can be affected by the pH of the environment.

So let's look at that in a little bit more detail.

So at the optimum pH, the enzyme has the perfect shape.

All of those bonds are functioning correctly, they're keeping the enzyme in the correct shape, and therefore the active site has its correct shape and it means that all of the active sites can be used properly and therefore they are full with substrate and the enzyme is essentially working as fast as it possibly can.

But as we move away from that optimum pH, the pH starts to interfere with some of those bonds which are holding the enzyme together.

So at other pHs, the enzymes bonds get disrupted, they get broken, and therefore the shape changes.

Now the breaking of those bonds is called denaturing, and this reduces the ability for the enzyme to catalyse the chemical reaction and therefore the rate decreases as well.

And we can see that in the graph.

We can see that's what's happening.

And if you look at those little diagrams of the enzymes on the screen, you can see the proper shape of the enzyme and how it's been denatured at pHs, which are outside of the optimum.

Now, if we carry on changing the pH away from the optimum into much more extreme pHs for the enzyme, the rate will ultimately reduce to zero because the more and more of the bonds will be broken within the enzyme and ultimately the enzyme will be fully denatured.

Essentially, it's reached a shape that it can now no longer catalyse any of the reactions because the active site no longer functions as an active site.

The shape has completely disappeared, and this is where the rate reduces to zero, no reaction can be catalysed, and therefore there is no reaction happening whatsoever.

So you can see that in the shape of the graph, and you can see that as we're moving away from the optimum pH, that is what is happening, the enzyme is being denatured.

Now that optimum pH will be a different pH depending on what type of enzyme is it.

It is whereabouts, what kind of conditions it normally operates in.

So cellular enzymes which are operating at a pH of about seven, then their optimum will be about seven.

But enzymes which are operating within the stomach acid for instance, will have a much different optimum pH.

For instance, those in operating in the stomach acid will have an optimum pH of about one or two compared to those in the small intestine, for instance, where their optimum pH will be more like eight.

So you can see that the pH optimum will change depending on what the enzyme is, but the same process will apply that as you move away from that optimum pH in either direction of pH, the enzyme activity will fall away because it will become denatured.

So let's just review that, make sure we've got our ideas straight in our head.

So we've got three students here, Laura, Izzy, and Jun, who are like trying to explain the effect of the pH on enzyme activity, but who is correct and who is not.

So Laura says the enzyme's active site changes shape in extreme pHs because bonds break.

Izzy says the enzyme has the best shape at the optimum pH and can work really fast.

And Jen says the enzyme denatures at the optimum pH because the active site is special shape.

So who is correct, who is incorrect? I'll give you five seconds to decide.

Okay, so considering Laura's explanation, she is correct, the enzymes ACTO site changes shape in extreme pHs because bonds break.

Let's look at Izzy now.

So she's also correct because the enzyme does have the best shape at the optimum pH and therefore can work really fast.

And let's finally look at Jun.

So he's incorrect because the enzyme doesn't denature at the optimum pH.

That is where it's working fastest.

But he is right in saying the active site is a special shape.

That part of his statement is correct.

So did you draw the same conclusions? Well done, well done if you did.

So what I'd like you to do now is to summarise all of that information by firstly describing what is meant by the enzyme's optimum pH.

Then I'd like you to describe how you can work out the optimum pH for an enzyme, then have a go at explaining how extreme pHs affect the enzyme and impact on the rate of reaction.

Now, you might wish to join all of those three tasks up into one longer paragraph rather than answering them step by step.

That's up to you.

And then finally, I'd like you to consider that some food is preserved in vinegar like pickled onions, preserved bee root, those sorts of things.

Now, vinegar has a low pH.

So what do you think that might do to the bacterial and fungal cells that might be trying to live on the preserved food on the pickled onions or the pickled beetroot? And therefore suggest why it is suitable method for preserving food, for some food anyway.

So pause the video, consider all of these questions in detail and come back to me when you are ready.

Okay, so let's see what you might have written.

So when I asked you to describe what is meant by the enzyme's optimum pH, you should have said that the optimum pH is the pH at which the reaction rate is highest or fastest.

When I asked you to describe how you can work out the optimum pH for an enzyme, you should have said that you would measure the time taken for the substrate to be converted into product at different pHs, then convert the time taken into rate by using one divided by time taken, and the pH, which the time taken is shortest, or the rate calculation is highest, indicates the optimum pH.

So just review your answer before we move on.

Then I asked you to explain how extreme pHs affect the enzyme and impact on the rate of reaction.

So you should have said that at extreme pHs, the bonds holding the enzymes together are broken.

This means that the enzymes shape changes and we call this process denaturing.

Ultimately, this reduces the ability of the enzyme to catalyse the reaction, and eventually the rate will reduce to zero as the enzymes are fully denatured.

So again, just review your answer against these points and make sure you've got all the correct parts.

Then finally, I gave you this scenario about preserving food in vinegar, which has a low pH, and asked you to suggest what this might do to the bacterial and the fungal cells, which are trying to live on those preserved foods, and therefore why preserving in vinegar is a suitable method for preservation of certain foods.

So you might have included ideas that bacterial and fungal cells have enzymes, and therefore the vinegar is an extreme pH for those enzymes.

Now, this means that the vinegar will denature the enzymes by breaking the bonds that hold the enzyme together in its specific shape, and therefore this changes the shape of the active site and the enzymes can no longer catalyse the reaction.

Now, this prevents the food from being decomposed by the bacteria or the fungi, and therefore it preserves it.

So just review your answer against mine and check that you've got all the really important points within your answer.

And well done.

That was a lot of really quite complex thinking.

So well done for having a go and for what you got, right as well.

Okay, so in our lesson today, we have seen that the rate of the reaction can be calculated by dividing one by the time taken to reach the end point of the reaction.

And we can plot rate on a graph against pH and use that to consider whether the data increases, or decreases our confidence in our original prediction.

Now, we've also seen that the enzyme reaction rate is highest at the optimum pH because all of the active sites are full and those active sites are of the right shape.

But at pHs above and below the optimum, the active site becomes denatured because the enzyme bonds that are holding it together in its special shape are broken.

So I hope you enjoyed our lesson today.

Thank you very much for joining me, and I hope to see you again soon, bye.