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 oven enzyme. Now, before we talk directly about the specificity constant oven 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 that are really, really high because they're able to determine value such as the V max and the K cat, or the maximal catalytic 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 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 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 K cat that we talked about in some of our previous lesson videos, um, which Onley 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 Maura 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 K cat or the turnover number is not always the best measure of catalytic efficiency, especially since it on Lee 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 K cat to the K M. And that's exactly what we're saying. Down below is that the specificity constant is this ratio of the K cat over the K M. And so this specificity constant ratio of the kick out over the K M is really what determines an enzymes 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 and 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 are at 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 Catholic efficiency indicated by the K cat alone. And so, under saturating substrate concentrations, the Que cat alone is what dictates the preference that enzyme has for the substrate. However, when the substrate concentrations are at non saturating levels or low substrate concentrations, then the catalytic efficiency is actually determined by not the K cat alone, but instead the specificity constant, which is the ratio of the K cat over the K M. 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 Kimo trips and which is a pep today's 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 kinda trips and has a preference for which amino acids it recognizes for cleavage and the pneumonic that helped us memorize that preference was free. Your worries like May, where we said that Kimo trips and has a preference for the aromatic amino acids, phenylalanine, tyrosine and trip to fan. And it has less of a preference for losing and Matheny. And so in our next video, we're going to talk about exactly how Kim a trip sends preference, relates directly to the specificity constant ratio. And so again, we're going to retouch on this idea here in our next lesson video. So hang on tight for this bullet point here. Now what I want you guys to note is that this ratio, the specificity, constant ratio of the cake cat over the K M 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 K cat as well as an enzymes affinity for its substrate, which is indicated by the K M. Now, larger ratios of this specificity constant represent mawr 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 I also want to note that this specificity constant ratio of the cake cat over the K M actually does have a maximum value that's approximately equal to 10 to the ninth with units of inverse polarity, inverse seconds. And we're going to touch mawr 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 K cat over K M. And in our next video, we're going to be able to, uh, talk even mawr about the specificity, constant ratio and how it applies to Kimo Trip sins preference. And so I'll see you guys in that video
3
concept
Specificity Constant
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12m
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all right. 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 on three different enzymes your ears, penicillin, eights and Kimo trips and which were already familiar with from our previous lesson videos. And so notice that in this first column right here were given the catalytic constants or the K cat or the turnover number and units of inverse seconds for all of these enzymes in their substrates and noticed that the K cat or the turnover number is on Lee going to occur under saturating substrate concentrations. And ultimately, that is what makes the K cat the maximal or the max catalytic efficiency that Onley occurs under saturating substrate. Concentrations now notice looking at this value of 10,000 just to refresh our memories on what exactly that means. Ah, value of 10,000 here means that just one molecule of the enzyme, Yuri's 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 and read as fast and down arrows and blue as slow. And so again, 10,000 here is pretty fast, especially in comparison to these other values. So for that reason, we could go ahead and give it five up arrows right here. Now moving on to this next column right here. Notice what we have is the McHale is constant or the k M and units of molar ity for all of these substrates and so ultimately, the K M we know is just a measure of an enzymes affinity. It's binding affinity for its substrate. And so the larger the K M 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 assay again up arrows and red meaning strong and down arrows and blue meaning weak. And so notice. Looking at this value here for year years that it has a value raise, uh, to the negative to tend to the negative, too. And ultimately that makes this number here in comparison to all of these other numbers the greatest or the largest number, uh, in comparison to these other values and recall that the larger the value of the K m, the weaker the affinity will be. And so because this is the largest value, we can say that it has a week affinity, and we could give it to 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 K M. Essentially, the ratio of this column over, uh, 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 two down arrows will cancel out with these two up arrows and we're just left with three up arrows. And so over here, next to the specificity constant, we can put three up arrows for the enzyme. Yuris, now moving on to penicillin, is here. Notice that it's catalytic constant of 2000 is much smaller than the catalytic constant of 10,000, but still penicillin. It's with a value of 2000 Can. One molecule of the enzyme penicillin, it's under saturating substrate. Concentrations can convert 2000 molecules of substrate into product per second. So that's still pretty fast. Not quite as fast as here he is. But we can go ahead and give it three up arrows here and then taking a look at its K M. Noticed that the it's raised to 10 to the negative fifth here and in comparison toe all of these other numbers. Uh, this number right here is actually the smallest number, and the smaller the K M value is, the stronger the affinity is. And so we can say that penicillin is has the strongest affinity for its substrate. So in this column right here, we can indicate the strength of the affinity by giving it two up arrows. So ultimately and the catalytic constant, uh, in order to get the arrows for penicillin is, all we need to do is some of these arrows. So the three up arrows, plus the two up arrows give us five up arrows so we could go ahead and indicate five up arrows here and then moving on. Knows what we have is chemo trips in with ah specific set of substrates which are female Alan entire scene and trip to fan. And we already know from our previous lesson videos that comma trips and has a preference for which enzymes it recognizes for cleavage and the pneumonic that helps us memorize kinda trip sins. Uh, preference is free. Your worries like May, where we know that kinda trips and has a preference for the aromatic amino acids Final Alan entire scene and trip to fan. And it has less of a preference for the amino acids losing and Matheny. And so for the aromatic amino acids phenylalanine, entire scene and trip to fan. We could say that these are going to be mawr preferred just based off of our pneumonic. And of course, we know that Lucy and Emma thinning here are going to be less preferred just based off off of our pneumonic. And we also know that an amino acids such as lysine is not going to be recognized by comma trips. And it also this substrate here is not preferred at all. And so, working with just kinda trip sins, Uh, substrates of female island, entire scene and trip to fan. Uh, if we were to convert this 100 here into a catalytic speed, we can see that it's much less than the penicillin is. And so it definitely needs toe have less up arrows. And so we'll go ahead and give it to up arrows Here Now, looking at the K M for, uh, this group of substrates here notice that it's raised to 10 to the negative fourth here and eso are these other three K m values as well. And so even though they do varied by just a little bit because they're all to tend to the negative fourth, we'll go ahead and assume that they all pretty much have the same km 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 two up arrows plus one up arrow. So that gives us three up arrows over here, and then we can move on to kinda trips and and Lucy and Emma thigh any. And so, uh, in comparison, uh, comparing the 0.63 catalytic constant to 100 it's way, way less so definitely there's, uh we can see that it's much, much slower when it is working with Lucy and and Matheny. But in comparison to listen, which is not preferred at all 0.63 is actually much greater. So for that reason, we could go ahead and give 0.63 just one up arrow here. And then, of course, 0.2 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 two up arrows here for the specificity constant. And then this one down arrow will cancel out with one up arrow and we'll end up getting zero up arrows over here. So we'll just put a zero over here just as that feller. 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 enzymes 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 enzymes preference for its substrate, just like the specificity, constant ratio. And so there is a difference between these two. And the difference is that, uh, the catalytic constant Onley determines and enzymes preference for substrate under saturating substrate concentrations, whereas the specificity constant will determine an enzymes preference for its substrate at non saturating or low substrate concentrations. And so, ultimately and enzymes, 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 Ah, very, very high speed does not necessarily mean that it's gonna have a high catalytic efficiency at low substrate concentrations. And so you can see that even though penicillin ACE has a lower, maximal catalytic efficiency, uh, with less up arrows, it ends up having a higher catalytic efficiency at lower substrate concentrations. And that's because the penicillin is has a greater binding affinity to its substrate than your ears does. And so, moving onto this next category here noticed that female Alan entire scene and trip to fan are more preferred at saturating substrate concentrations. Uh, because it has a higher catalytic efficiency on ah higher. I'm sorry. Maximal catalytic efficiency and female Alan Entire scene and trip to fans are also Mawr preferred under low substrate concentrations because it has a higher specificity constant, and we can see how losing and meth I inning are definitely less preferred at saturating substrate concentrations because they have one up arrow here and you can see that they're definitely less preferred, uh, than fetal Alan entire scene in trip to fan at low substrate concentrations, but still mawr preferred than, uh, amino acids that are not preferred at all. And so ultimately here, what we can see is that, uh, the specificity constant ratio is a balance of the K cat 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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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 K cat over K M 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 cake at over K M. Not only is it diffusion controlled limited, but it's also limited by the rate constant K one, 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 on Lee 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 K one and the specificity constant of K cat over km are going to be equal to the maximum rate of diffusion in water, which is going to be 10 to the ninth. Inverse polarity, 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 note is that the free enzyme and the free substrate can on Lee associate with each other via diffusion. And so what this means is that this rate constant here of K one is going to be limited by the maximum rate of diffusion. And so the K one value cannot be any greater than the maximum rate of diffusion. And also because we know from our previous lesson videos that the McHale is constant. K M is expressed as the ratio of the some of the enzyme substrate complex dissociation rate constants, or K minus one plus K two over the association rate constant K one. Because K one is included in the K M. What this means is that the K M is also going to be limited by the diffusion, uh, the max rate of diffusion and because the K M is included into the specificity constant, which is the ratio of the cake cat over the K. M. Uh, that means that the specificity constant is also going to be limited by diffusion. So up above notice. What we said is that the rate constant K 13 k m and the specificity constant of cake cat over cam are all directly limited by the maximum rate of diffusion in water, which again is 10 to the ninth. Inverse morality inverse seconds, just as we set up above. And so what's interesting to note here is that an enzyme whose, uh, specificity constant ratio is actually equal to this diffusion controlled max value that we indicated up above 10 to the ninth inverse similarity in verse seconds is actually referred to as a catalytic lee perfect enzyme. And so a catalytic lee perfect enzyme is essentially an enzyme who, upon the substrate binding to the enzyme, uh, the enzyme will immediately and almost instantaneously convert the substrate into product. And so that's why were we refer to these enzymes as Catalytic Lee 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 ninth inverse similarity in verse seconds, and this maximum value is a diffusion controlled limit. And so this year concludes our lesson, and we'll be able to get some practice utilizing these concepts, moving forward in our practice problems, 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.
