Specificity Constant - Video Tutorials & Practice Problems
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Specificity Constant
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In this video, we're going to begin our discussions on the specificity constant of an enzyme. Now before we talk directly about the specificity constant of an enzyme, it's first important to recognize a few key points. And the first key point to recognize is that when biochemists are studying or characterizing enzymes in a laboratory setting, it definitely is useful for them to study those enzymes in the lab under saturating substrate concentrations efficiency under saturating substrate concentrations. However, what's also really important to know is that the substrate concentrations within a cell or within a biological system are not always saturating. And so what this means is that saturating substrate concentrations is not always the best way to study enzymes. And again, this has to do with the fact that under physiological conditions or conditions within a cell, again the substrate concentrations are not always saturating, and instead the tendency is for the cell to maintain substrate concentrations approximately equal to the k m of the enzyme. Also, another reason for why it's not always the best to study enzymes at saturating substrate concentrations is because under saturating substrate concentrations, that does not allow the biochemist to account for the binding affinity that an enzyme has for its substrate or the enzyme substrate binding affinity cannot be taken into account under saturating substrate concentrations. And that is because, regardless of the binding affinity that an enzyme has for its substrate, even if the binding affinity is really really really low, under saturating substrate concentrations, all of the enzyme is going to be associated with the substrate to form the enzyme substrate complex. And so, again, that will not allow the biochemist to account for the binding affinity that an enzyme has for its substrate under saturating substrate concentrations. And so, again, what this really all means is that the maximal catalytic efficiency or the kcat that we talked about in some of our previous lesson videos, which only occurs under saturating substrate concentrations, is not always going to be the most relevant measure of the catalytic efficiency of an enzyme. And instead, a better overall measure for the catalytic efficiency of an enzyme is the specificity constant. And so we're going to define and talk more about the specificity constant in our next lesson video. So I'll see you guys there.
2
concept
Specificity Constant
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So in our last lesson video, we said that the maximal catalytic efficiency of an enzyme indicated by the catalytic constant kcat or the turnover number is not always the best measure of catalytic efficiency, especially since it only occurs under saturating substrate concentrations and saturating substrate concentrations are not always relevant, especially inside of a cell. And so in this lesson video, we're going to talk about another better overall measure of catalytic efficiency, specifically under non saturating substrate concentrations or low substrate concentrations, and that's exactly where the specificity constant comes into play. And that's because, as you guys may have already determined, the specificity constant is this ratio of the kcat to the k m, and that's exactly what we're saying down below, is that the specificity constant is this ratio of the kcat over the kilometers. And so this specificity constant ratio of the kcat over the kilometers is really what determines an enzyme's preference for a substrate, specifically at non saturating substrate concentrations or low substrate concentrations. And so it turns out that substrate preference or the preference that an enzyme has for a particular substrate, is determined by the catalytic efficiency. And so the higher the catalytic efficiency, the greater the preference an enzyme has for that substrate. Now, it also turns out that catalytic efficiency actually depends on the concentration of substrate and whether or not those substrate concentrations saturating levels or non saturating levels. Now, if the substrate concentrations are at saturating levels, then that means that the catalytic efficiency is going to be determined by the maximal catalytic efficiency indicated by the kcat alone. And so under saturating substrate concentrations, the kcat alone is what dictates the preference that an enzyme has for the substrate. However, when the substrate concentrations are at nonsatrating levels or low substrate concentrations, then the catalytic efficiency is actually determined by not the kcat alone, but instead the specificity constant, which is the ratio of the kcat over the kilometers. And that means that the specificity constant ratio is what determines the preference that an enzyme has for its substrate at non saturating or low substrate concentrations. Now, what I want you guys to recall from way back in our previous lesson videos is that we talked about this enzyme called chymotrypsin, which is a peptidase that recognizes specific amino acids to cleave peptide bonds between them. And so what I, want you guys to recall is that we said that chymotrypsin has a preference for which amino acids it recognizes for cleavage, and the mnemonic that helped us memorize that preference was Free Your Worries, Like Me, where we said that chymotrypsin has a preference for the aromatic amino acids Phenylalanine, Tyrosine, and tryptophan, and it has less of a preference for Leucine and methionine. And so in our next video, we're going to talk about exactly how chymotrypsin's preference And so, again, we're going to retouch on this idea here in our next lesson video. So hang on tight for this bill bullet point here. Now, what I want you guys to note is that this ratio, the specificity constant ratio of the kcat over the kilometers, is just another measure of catalytic efficiency. And specifically, the specificity constant ratio is the catalytic efficiency when an enzyme is not saturated with substrate or essentially at low substrate concentrations. Now this specificity constant ratio accounts for both the maximal catalytic efficiency kcat as well as an enzyme's affinity for its substrate, which is indicated by the k m. Now larger ratios of this specificity constant represent more efficient enzymes, and, of course, more efficient enzymes, as we said earlier, is going to indicate a higher preference for the substrate at lower substrate concentrations. And, I also want to note that this specificity constant ratio of the kcat over the kilometers actually does have a maximum value that's approximately equal to 10 to the 9th with units of inverse molarity inverse seconds. And we're going to touch more on this idea later in our course, so also hang on for this bullet point as well until later in our course. So this here concludes our introduction to the specificity constant ratio of the kcat over kilometers, and in our next video, we're going to be able to talk even more about the specificity constant ratio and how it applies to chymotrypsin's preference. And so I'll see you guys in that video.
3
concept
Specificity Constant
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12m
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Video transcript
Alright. So here we have a table that looks a lot more complicated than what it actually is, and all it's really giving us is information 3 different enzymes, urease, penicillinase, and chymotrypsin, which we're already familiar with from our previous lesson videos. And so notice that in this first column right here, we're given the catalytic constants or the kcat or the turnover number in units of inverse seconds for all of these enzymes and their substrates. And notice that the kcat or the turnover number is only going to occur under saturating substrate concentrations. And, ultimately, that is what makes the k cat the maximal or the max catalytic efficiency that only occurs under saturating substrate concentrations. Now, notice looking at this value of 10,000 1,000, just to refresh our memories on what exactly that means. A value of 10,000 here means that just one molecule of the enzyme urease, specifically under saturating substrate concentrations, can convert 10,000 molecules of substrate into product per second. So that is incredibly fast, especially in comparison to all of these other values that we have here. So, for that reason, what we can do is we can indicate the catalytic constant speed in this column, by putting up arrows in red as fast and down arrows in blue as slow. And so, again, 10,000 here is pretty fast, especially in comparison to these other values. So for that reason we can go ahead and give it 5 up arrows right here. Now, moving on to this next column right here, notice what we have is the Michaelis constant or the Kilometers in units of molarity for all of these substrates. And so, ultimately, the kilometers we know is just a measure of an enzyme's affinity, its binding affinity for its substrate. And so the larger the Kilometers value is, the weaker the affinity will be. And so over here in this column, what we can do is indicate the enzyme affinity for the substrate, as, again, up arrows in red meaning strong and down arrows in blue meaning weak. And so notice looking at this value here for Euryase that it has a value, raised to the negative 2, 10 to the negative 2. And, ultimately, that makes this number here, in comparison to all of these other numbers the greatest or the largest number in comparison to these other values. And recall that the larger the value of the kilometers, the weaker the affinity will be. And so because this is the largest value, we can say that it has a weak affinity and we can give it 2 down arrows here. And then of course, in this final column, what we have is the specificity constant ratio, which we introduced in our last lesson video. And so this is the ratio of the k cat over the kilometers, essentially the ratio of this column over this column right here. And so, ultimately, what we're going to do is we're going to take the ratio of the arrows of this column and this column right here so that we can also get arrows over here for the specificity constant. And so, ultimately, down arrows are going to cancel out with up arrows, and so these 2 down arrows will cancel out with these 2 up arrows and we're just left with 3 up arrows. And so over here, next to the specificity constant, we can put 3 up arrows for the enzyme, urease. Now, moving on to penicillinase here, notice that its catalytic constant of 2,000 is much smaller than the catalytic constant of 10,000. But still, penicillinase with a value of 2,000 1,000 molecules of substrate into product per second. So that's still pretty fast, not quite as fast as urease, but we can go ahead and give it 3 up arrows here. And then, taking a look at its kilometers, notice that the, it's raised to 10 to the negative 5th here. And in comparison to all of these other numbers, this number right here is actually the smallest number. And the smaller the kilometers value is, the stronger the affinity is. And so we can say that penicillinase has the strongest affinity for its substrate. So in this column right here, we can indicate, the strength of the affinity by giving it 2 up arrows. So, ultimately, in the catalytic constant, in order to get the arrows for, penicillinase, all we need to do is sum up these arrows. So the 3 up arrows plus the 2 up arrows give us 5 up arrows. So we can go ahead and indicate 5 up arrows here. And then moving on, notice what we have is chymotrypsin, with a specific set of substrates, which are phenylalanine, tyrosine, and tryptophan. And we already know from our previous lesson videos that chymotrypsin has a preference for which enzymes it recognizes for cleavage. And the mnemonic that helps us memorize chymotrypsin's preference is free your worries like me, where we know that has a preference for the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, and it has less of a preference for the amino acids leucine and methionine. And so for the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, we can say that these are going to be more preferred just based off of our mnemonic. And of course, we know that leucine and methionine here are going to be less preferred, just based off of off of our mnemonic. And we also know that an, amino acid such as lysine is not going to be recognized by chymotrypsin at all. So this substrate here is not preferred at all. And so, working with just, chymotrypsin's, substrates of Phenylalanine, Tyrosine, and tryptophan, if we were to convert this 100 here into a catalytic speed, we can see that it's much less than the penicillinase. And so, it definitely needs to have less, up arrows. And so, we'll go ahead and give it 2 up arrows here. Now looking at the, kilometers for this group of substrates here, notice that it's raised to 10 to the negative 4th here. And, so are these other 3 kilometers values as well. And so even though they do vary by just a little bit because they're all to, 10 to the negative 4th, we'll go ahead and assume that they all pretty much have the same value, and we'll go ahead and give them all one up arrow here for the strength of the affinity. And so ultimately what we can get over here for this set of substrates is that, we have 2 up arrows plus 1 up arrow, so that gives us 3 up arrows over here. And then, we can move on to chymotrypsin and leucine and methionine. And so, in comparison, comparing the 0.63 catalytic constant to 100, it's way way less. So definitely, there's, we can see that it's much much slower when it is, working with leucine and methionine. But in comparison to lysine, which is not preferred at all, 0.63 63 just one up arrow here, and then, of course, 0.02 is going to be a down arrow, very, very slow. And so ultimately, if we sum up these arrows, one up arrow here plus one up arrow here is gonna give us 2 up arrows here for the specificity constant. And then this one down arrow will cancel out with the one up arrow and we'll end up getting 0 up arrows over here. Here. So we'll just put a 0 over here just as that filler. And so, ultimately, what you can see is that these arrows here give us a relative visual of the value of the specificity constant. And so, ultimately, what you can see is that, from our previous lesson videos, we said that the specificity constant was a measure of the catalytic efficiency specifically at non saturating or low substrate concentrations. And we also said that the specificity constant ratio would determine the enzyme's preference for the substrate, but specifically at low or non saturating substrate concentrations. But notice over here that the catalytic constant is also, determining an enzyme's preference for its substrate just like the specificity constant ratio. And so there is a difference between these two, and the difference is that, the catalytic constant only determines an enzyme's preference for substrate under saturating substrate concentrations, whereas the specificity constant will determine an enzyme's preference for its substrate at nonsatrating or low substrate concentrations. And so, ultimately, an enzyme's preference for its substrate is going to depend specifically on the levels of substrate concentration and whether or not the substrate concentrations are saturating or if the substrate concentrations are non saturating or low substrate concentrations. And so, ultimately, what we can see looking at this row right here, notice that just because it has a high catalytic efficiency, a very very high speed, speed, does not necessarily mean that it's going to have a high catalytic efficiency at low substrate concentrations. And so, you can see that even though penicillinase has a lower maximal catalytic efficiency, with less up arrows, it ends up having a higher catalytic efficiency at lower substrate concentrations, and that's because the penicillinase has a greater binding affinity to its substrate than urease does. And so, moving on to this next category here, notice that phenylalanine, tyrosine, and tryptophan are more preferred at saturating substrate concentrations, because it has a higher catalytic efficiency, and a higher, I'm sorry, maximal catalytic efficiency, and phenylalanine, tyrosine, and tryptophan are also more preferred, And, Phenylalanine, Tyrosine, and Tryptophan are also more preferred, under low substrate concentrations because it has a higher specificity constant. And, we can see how leucine and methionine are definitely less preferred at saturating substrate concentrations, because they have, one up arrow here. And, you can see that they are definitely less preferred than Phenylalanine, Tyrosine, and Tryptophan at low substrate concentrations, but still more preferred than amino acids that are not preferred at all. And so ultimately here, what we can see is that, the specificity constant ratio is a balance of the kcat and the k m. And we'll be able to apply a lot of these concepts that we've learned here moving forward in our practice problems. So I'll see you guys there.
4
Problem
Problem
Use the data in the chart below to provide answers to the following problems:
A) List the substrates from most preferred to least preferred under physiological conditions.
a) B, A, C. b) C, B, A. c) B, C, A. d) A, C, B.
B) List the substrates from most preferred to least preferred under saturating [S].
a) B, A, C. b) C, B, A. c) B, C, A. d) A, C, B.
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5
concept
Specificity Constant
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So in our previous lesson videos on specificity constant, we briefly mentioned that the specificity constant ratio of kcat over kilometers actually has a maximum value, and it cannot be higher than that maximum value. And so it turns out that this limit to the maximum value of the specificity constant is actually diffusion controlled. And so, the maximum value of this specificity constant of kcat over Kilometers, not only is it diffusion controlled, limited, but it's also limited by the rate constant K 1, which we know is the free enzyme and the free substrate association rate constant. And so, this is because the free enzyme and the free substrate association will actually occur via diffusion. And so what this means is that the free enzyme and the free substrate can only associate with each other to form the enzyme substrate complex as fast as the maximum rate of diffusion in the solvent. And of course, in biological systems, the solvent is going to be water, and so therefore we can say that the maximum values of both the rate constant k1 and the specificity constant of kcat over Kilometers are going to be equal to the maximum rate of diffusion in water, which is going to be 10 to the 9th inverse molarity inverse seconds. And so down below on the left hand side over here, what we have is our typical enzyme catalyzed reaction. And so all I want you guys to know is that the free enzyme and the free substrate can only associate with each other via diffusion. And so what this means is that this rate constant here of k 1 is going to be limited by the maximum rate of diffusion. And so the k1 value cannot be any greater than the maximum rate of diffusion. And, also because we know from our previous lesson videos that the Michaelis constant kilometers is expressed as the ratio of the sum of the enzyme substrate complex dissociation rate constants, or k minus 1 plus k 2, over the, association rate constant k one. Because k one is included in the Kilometers, what this means is that the Kilometers is also going to be limited by the diffusion, the max rate of diffusion. And, because the kilometers is included into the specificity constant, which is the ratio of the k cat over the k m, that means that this specificity constant is also going to be limited by diffusion. So up above, notice what we said is that, the rate constant k one, the kilometers, and the specificity constant of kcat over kilometers are all directly limited by the maximum rate of diffusion in water, which again is 10 to the 9th, inverse molarity, inverse seconds, just as we said up above. And so what's interesting to note here is that an enzyme whose, specificity constant ratio is actually equal to this diffusion controlled max value that we indicated up above, 10 to the 9th inverse molarity inverse seconds, is actually referred to as a catalytically perfect enzyme. And so a catalytically perfect enzyme is essentially an enzyme who, upon the substrate binding to the enzyme, the enzyme will immediately and almost instantaneously convert the substrate into product. And so, that's why we're, we refer to these enzymes as catalytically perfect. And so, it's important to note here that the specificity constant ratio does have a maximum value, and it's going to be 10 to the 9th inverse molarity inverse seconds, and this maximum value is a diffusion controlled limit. And so this here concludes our lesson, and, we'll be able to get some practice utilizing these concepts moving forward in our practice problem. So I'll see you guys there.
6
Problem
Problem
Which of the following options is correct concerning the turnover number (k cat) and the specificity constant?
A
kcat reveals how well an enzyme works & its preference for S.
B
Specificity constant is defined as (kcat)(Km).
C
A large kcat indicates a less efficient enzyme.
D
kcat = Vmax/[ES].
E
Specificity constant is defined as Km / kcat.
F
A small Km indicates a more efficient enzyme.
7
Problem
Problem
Use the Lineweaver-Burk plot to help you calculate the Vmax, kcat, Km and specificity constant for the enzyme.
Assume the [E]T = 2.9 nM. Hint: Pay close attention to units.
Vmax = ___________.
kcat = ___________.
Km = ___________.
kcat / Km = ___________.
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Problem
Problem
Explain the steps you could take to accurately find the K m, Vmax, and specificity constant for an enzyme from the following kinetic data, assuming the experiments were all done with [E]T = 0.1 mM.
The specificity constant is obtained at low [S] via variable substitution into the Michaelis-Menten equation (Vmax = kcat[E]T). Considering this about the MM-equation, what is the relationship between changes in [S] & V0 when the [S] is super small and well below the Km?
A
The [S] term cancels out completely in this equation, so there is no effect of changing substrate concentration.
B
The [S] term in the numerator is negligible, so there is no impact of changing substrate concentration.
C
Because the enzyme has reached Vmax, there is no effect of changing substrate concentrations on enzyme velocity.
D
[S] term in the denominator is negligible compared to Km, so the relationship between [S] & V0 is directly proportional.