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Biochemistry

Learn the toughest concepts covered in Biochemistry with step-by-step video tutorials and practice problems by world-class tutors

8. Protein Function

Chymotrypsin's Catalytic Mechanism

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Chymotrypsin's Catalytic Mechanism

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in this video, we're going to begin our discussion on chemo trip sins, Catalytic mechanism. So, Kim, a trip sins catalytic mechanism can actually be broken up into two separate phases. The first phase is referred to as the ace elation phase, and the second phase is referred to as the D. A s elation phase. And so notice that we have both of these phases color coded down below, where the yellow background here represents the isolation phase. And then, of course, the blue background over here represents the D. A s elation phase and so moving forward in our course, we're going to talk about each of these two phases in ah, lot more detail. Now, notice that in the ace elation phase, we're starting off with this yellow half circle here that represents chemo trip sins active site, which we know has the catalytic triad. However, we're only showing the Syrian amino acid residue of the catalytic triad in this particular image. And we know that the catalytic triad well, actually establish this searing as a stronger nuclear file, which is why it has this negative charge on it and so noticed that kinda trips is all about cleaving specific peptide bonds. Uh, more specifically, it cleaves the C terminal peptide bonds of aromatic amino acid residues. And so here in pink, what we have is a peptide bond that's going to be cleaved by Kimo trips and notice that in the first phase the isolation phase here, uh, which will notice is that part of our substrate here is going to be cleaved off. But the other part of our substrate is going to be attached to the kind of trips and active site CO violently. And so there is some CO Vaillant catalytic mechanism in this, uh, mechanism that we'll talk about more in our next video. However, what I want you guys to notice is that in the isolation phase, this peptide bond is being cleaved. So at the end of the ace elation phase, the peptide bond of interest that we were trying to cleave has already been cleaved. However, Theis elation phase has Aysal ated are enzymes. So now we have an Aysal enzyme here that is co violently modified. So in the d. A s elation phase, it's all about regenerating the original enzyme so that the mechanism can occur again and so notice that water will come into play here. Toe hide relies this bond and release this portion of the substrate and regenerate our original ends. I'm here and this hydrogen again. It can be removed through the catalytic triad to establish steering once again as a strong nuclear file, as it was before. And so again, we'll be able to talk more and more details about each of these two phases as we move forward in our course. And in our next video, we'll talk the details about the isolation phase, so I'll see you guys in that video.
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Chymotrypsin's Catalytic Mechanism

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in this video, we're going to talk about Kim a trip sins first phase of its catalytic mechanism, which is the isolation phase. And so chemo trip sins Ace elation phase can really be broken down into four general steps that will talk about down below, but really the main take away of kinda trip since four step ace elation phases right here in this wine. And that is that Covalin catalysis is going to form an Esther linkage that will co violently link kinda trips and active site with the substrate as we'll see down below. But it turns out that this Covalin Esther linkage that forms in the isolation phase is really just a temporary covalin linkage. Because in the second phase, the D. A s elation phase that we'll talk about in our next video this co violent language is going to be broken. However, in addition to this Esther linkage that is created in the isolation phase Thebe peptide bond of interest, that kinda trips and is supposed to be cleaving is actually going to be broken or cleaved. And so notice that ace elation phase is really just a nuclear Filic Aysal substitution, which is a catalytic mechanism that you guys probably covered in your previous organic chemistry courses. And again, Theus Elation phase can really be broken down into these four general steps, which are listed down below. And they are substrate binding nuclear filic attack, removal of leaving group or the LG for short and end of the face and notice that down below in our image, the numbers that we have down below correspond with numbers that we have up above. And so, starting with the first phase, it is substrate binding, which really speaks for itself. So the substrate of chemo trips and is going to be the peptide that's going to be cleaved. And so the peptide that's going to be cleaved is going to bind to chemo trips and active site. And so if we take a look down below, it are image over here on the far left hand side. What we have is chemo trip sins Active site, which has that catalytic triad that we talked about in our previous lesson videos, which are Asper Tate 102 History in 57 searing 95. And so we'll see these three amino acids throughout the entire catalytic mechanism. Now, which will notice is that in the very first step right here, the substrate is coming and binding to Kimo trip sins active site, which is down below. And so notice that this substrate is actually a peptide that has an amino end over here with an NH three plus. And it has a car box will end over here with a car box Late group and notice that we have these particular amino acids represented by these circles. So four total amino acids, allen in and veiling we know are non polar amino acids, but they're not aromatic, and then Argentine over here is a charged amino acid. But again, it's not aromatic. So the Onley aromatic amino acid here is going to be the F, which is final, a winning and that eyes going to be an amino acid that kinda trips and will recognize for cleavage because it's aromatic. And that's why we have this. A darker shade of purple toe show that this is one that kind of trips and is going to recognize for cleavage and, of course, common trips and is going to cleave the C terminal peptide bond so it's gonna cleave the peptide bond closest to the C terminal end, which is actually this peptide bond right here in pink. And so what you'll notice is that, uh, in our previous lesson video we said that history in 57 is going to hydrogen bond with both, uh, searing 1 95 and Asper Tate 102 And all of this hydrogen bonding is really just used to establish steering 1 95 as a strong nuclear file. And really, this leads us into our second step nuclear Filic attack. And so, since steering 1 95 has been established as a strong nuclear file, it's going to perform a nuclear filic attack on the carbonnel carbon of the substrate toe form an unstable Tetra Hydro intermediate. And so notice that history in 57 here is going to be acting as a base and essentially acquiring this hydrogen on Syrian and the electron density that was in this bond here is going to be used to attack perform a nuclear filled attack on the carbonnel carbon atom of the substrate. And then, of course, the electron density in this double bond here is going to shift up to the oxygen atom. And ultimately, what this does is it forms this tetra hydro intermediate that we see right here. And so here we can fill in Tetra Hydro Intermediate and this Tetra Hydro intermedia is actually fairly unstable with this blue background here. However, what's important to know is that a region uncommon trips and active site referred to as the oxy and I in whole eyes actually going to stabilize uh, the Tetra Hydro Intermediate and so we can see the oxy anti and whole right here, which is really just this yellow curve that we see and this is used to stabilize again this tetra hydro intermediate that we see to ensure that it can readily form now again, which will notice, is that this green arrow here is really Covalin. Ca Tallis is because the enzyme is forming a co violent Esther linkage right here with the substrate. And so this, uh, linkage right here because it is a co violent language. It is a newly formed Esther Covalin linkage that links kinda trips in Syrian 1 95 to the Carbonnel carbon of the substrate. And so this is our first type of, uh, co violent, uh, catalysis that we're going to see. This is the first type of catalysis that we're seeing Kind of trips and perform is co violent catalysis. So that leads us to our third step. And in the third step, we have removal of the leaving group, which is the LG here for short. And in this step, the Tetra Hydro Intermediate is going to collapse. History in 57 is going to act as an acid, and the peptide bond is going to be broken. All right, so if we take a look at our image down below again, here is our Tetra Hydro Intermediate. And this particular reaction arrow right here represents the collapse of the Tetra Hydro Intermediate. And you can see that the electron density here in this peptide bond is being used to perform a nuclear filic attack on this acidic hydrogen. And in the process, the peptide bond is going to be cleaved or broken. And so you can see that in step number three, the peptide bond indeed is being cleaved and broken. And when you look down below noticed that the peptide bond is no longer there, as it was up above. And of course, because history is acting as an acid to donate this acidic hydrogen, it's going to result in the conjugate base of the history, which does not have a charge anymore. And so this leads us to step number four, which is really just the end of the isolation face. So at this point, what we have is an enzyme that has been Aysal ated. Um, and the released aiming portion of the substrate is going to freely diffuse away. And so notice that, uh, here, what we have is the released, uh, aiming portion. And this portion of the substrate, as we'll see over here is really just going to defuse away in step number four. And the end result is we have kinda trips and active site that is covertly linked to the substrate via this Esther linkage here and together, this right here is forming the Aysal enzyme. This whole thing is referred to as the hassle pens on. And so, because at the end of the ace elation phase, we end with an Aysal enzyme. That's exactly where we're going to pick up with when we start the d. A s elation phase, which is going to remove this portion of the substrate and restore the original enzyme back up here. And so again, the main take away of chemo trip sins Ace Elation phase is really that Covalin Catalysis forms an Esther linkage that creates the Aysal enzyme, and the peptide bond of interest is actually going to be broken. And so that concludes this video and I'll see you guys in our next video where we'll talk about the D. A solution phase.
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Chymotrypsin's Catalytic Mechanism

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So now that we've covered the first phase of kinda trip since catalytic mechanism in this video, we're going to move on to the second phase of kinda trip since catalytic mechanism, which is the D. A s elation phase. Now, just like the isolation phase kinda trips. Ines de ace elation phase can be summed up in four general steps that will talk about down below now because the D. A s elation phase is really just a continuation of the isolation phase, we've continued the numbering system down below, starting with number five now really the main take away of kinda trip since four stepdad isolation phase is right here. And that is that the CO Vaillant Esther linkage that was formed in the ace elation phase is going to be hydrolyzed or broken in order to regenerate or restore the original camo trips in enzyme so that it can again perform the same exact catalytic mechanism once again on a different substrate. And so what will notice is that this d ace elation phase of kinda trip since catalytic mechanism is really just an Esther hydrolyzed station which recall from your previous organic chemistry courses is just a specific catalytic mechanism and notice that these steps that we have down below are very much similar and parallel to the steps from the isolation phase. So we still have substrate binding nuclear filic attack, removal of leaving group or LG for short and the end of the phase. And of course, the numbers that are down below in our image are going to correspond with numbers up above in the text. And so starting off with this first step here, what we have is substrate binding. And so in the d. A s elation phase, there's going to be a new substrate that's going to enter the kinda trips and active site. And that is going to be a water molecule. And H 20 molecule is going to come into play. And once this h 20 water molecule enters kind of trip sins active site, uh, the remainder of the mechanism is gonna be pretty much a Siris of very similar steps that are gonna repeat from the isolation phase. So what we'll see is down below. We have the Aysal enzyme that we left off with in the isolation phase and which will notice is that in the very, uh, the fifth step here, which is the first step of the D. A s elation phase a water molecule H 20 molecule is entering into the active site. And so, in the six step here, the nuclear Filic attack histamine 57 is going to act as a base to deep protein, ate that water molecule. And that's going to create a hydroxide ion that is going to act as a nuclear file and attack the carbonnel carbon atom of this portion of the substrate here and that is going toe once again create a tetra hydro intermediate. And so, if we take a look at our image down below, we can see the water molecule comes into play. Here is the water molecule and then history in 57 here is going to act as a base through general basic Attallah sis, and it's going to essentially pick up this hydrogen from the water molecule. And then the electron density that used to be in this bond is going to be used to attack the carbonnel carbon atom of this portion of the substrate here. And so this is going to generate a Tetra Hydro Intermediate once again And so this tetra hydro intermediate here eyes going to be unstable. However, once again we can say that the Oxy and I n hole, which is again a region and common trips into active site. It is going to stabilize the Tetra Hydro Intermediate in order to ensure that it can readily form. And so, in the seventh step here, what we have is removal of the leaving group. And so in order for that to happen, the Tetra Hydro Intermedia is going to collapse. Once again, his city and 57 is going to act as an acid again. And the Esther Bond is going to be broken. And so, if we take a look down below, notice that in the seventh step here, what happens is this Tetra Hydro Intermediate is going to collapse through this area arrow right here and then this history in 57 is going to act as an acid and donate this blue hydrogen here. And of course, the electron density here forming the Esther Bond is going to pick up that hydrogen. And, uh, that is going to cause this Esther linkage here to be broken And so that's exactly what we're showing here in the seventh step is that the Esther Bond is being cleaved. And so notice that we have the Esther, uh, bond up above here. However, over here it has been cleaved, and it's no longer present. And so in the eighth and final step, we have the end of the phase. And so at this point, the enzyme kinda trips and has been de asi dilated, and the released carb oxalic acid portion of the substrate is free to defuse away. And so we can see here we have this released carb oxalic acid portion and it is free to defuse away. And ultimately what we're left with is the original camo trips and enzyme, which is now restored back to its original state. And it's now ready for another round of Catalunya, sis. And so really, that is all of the chemo trips in catalytic mechanism. It's broken up into the isolation phase and the D. A s elation phase and the steps are very similar and very parallel. And so in our next video will be able to put together both the isolation and D C d ace elation phase, uh, in tow, one video so that we can look at Theo entire catalytic mechanism all at once. So that concludes this video, and I'll see you guys in our next one.
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Problem

Chymotrypsin’s acylation phase is below. The catalytic process of __________ is illustrated by arrow # ____ and the catalytic process of _________________________ is illustrated by arrow # ____.

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Chymotrypsin's Catalytic Mechanism

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in this video, we're going to do a recap of comma trip sins, entire catalytic mechanism, including both the isolation and D ace elation phases. And so notice down below in our image that we're not showing you any of the reaction mechanistic arrows. And that's because we're going to inter actively fill them in as we recollect the exact steps of each of the different phases. And so notice that down below the steps that have a yellow background here are correlating with the isolation phase. And then the steps that have a blue background are correlating with the D. A s elation phase. And so what you'll notice is over here in the far top left, we're showing you kinda trip since catalytic mechanism. Which, of course, has the catalytic triad, uh, which are the three amino acids Aspect 8102 history and 57 Syrian 95. And so in the first stage, the first step of the isolation phase, we have binding of the substrate. So here is our substrate, and it's going to be bound to the kind of trip since active site. Now in the second step, what we're going to have is a nuclear filic attack. And so recall that history in 57 here is going to be hydrogen bonding with Syrian 95 and aspect 810 to all to make history in 57 act as a better base so that it can make Syrian 1 95 a better nuclear file. And so, essentially, what's gonna happen is his 13. 57 here is going to act as a base and take up this hydrogen right here and then the extra electron density right here, um, is going to attack a nuclear filic attack on this carbonnel carbon atom. And then, of course, theologian on density here in this double bond is going to shift up onto the oxygen. And so recall that this green arrow here is going to be covalin catalysis because a new Covalin bond is forming between the enzymes active site as well with the substrate. And so in the second step, what we end up generating is a tetra hydro intermediate, which is what we have here and recall that the Tetra Hydro Intermediate is going to be unstable and so ah portion of comma trip Since active site referred to as the Oxy and I in whole is going to help stabilize the Tetra Hydro Intermediate that we have here. And of course, uh, the third step is going to be, uh, the removal of the leaving group. And so in this third step, essentially, what's going to happen is the Tetra Hydro Intermediate appear is going to collapse. History in 57 is going to act as an acid. And so it's going to essentially donate this hydrogen right here. And, uh, let's make this era little clear. And this hydrogen is gonna be donated and it's gonna be collected and picked up by the peptide bond here that's going to ultimately be broken. And so in the third step, we do have, uh, that the peptide bond is going to be cleaved now. Ultimately, what this results in is this free portion of the substrate is going to defuse away, and we're left with a Covalin attachment here to between the substrate and the enzyme. And so this is all referred to as the Aysal enzyme. And this is exactly where we pick up with when we start the D. A s elation phase and So notice that with the d A s elation phase again, we're going to repeat the similar steps. So we're gonna have introduction of a substrate. And so now we're introducing a new substrate water. So water molecule is gonna come into the active site and so we're gonna have a Siris of similar steps. And, uh, in step six, essentially, what's gonna happen is a nuclear filic attack, and so hissed 18 57 is again going to help establish a strong nuclear file. So it is going to act as a base and collect a hydrogen, um, Adam from the water molecule. And then this extra electron density here on the water molecule, uh, which would be generating a hydroxide iron, would be able to attack, perform a nuclear filic attack on the Carbonnel carbon atom again. And, of course, the electron density here on the double bond is gonna have to shift up. And so ultimately, this generates a Tetra Hydro intermediate that is relatively unstable but is stabilized through the Oxy and I in whole of chemo trips and active site. And so in the next step, what we have is removal off the leaving group and believing Group here is actually going to be the remainder of the substrate is gonna act as a leaving group. And so in this step, what needs to happen is the extra electron density here on the oxygen is going to shift down for the Tetra hydro collapse. And then the electron density here in this Esther Bond is going to be used to perform a nuclear filic attack on this acidic hydrogen right here. And then, of course, the electron density here is going to shift up onto the nitrogen. And in the process of all of this, this Esther Bond is being cleaved. And so that's exactly why we have Esther Bond being cleaved here. And you can see that up above the Esther Bond that used to be linking the substrate is no longer there because it was cleaved. And this allows the rest of our substrate here to defuse away. And in the process, it regenerates the original Kimo trips and active site that we had at the very beginning of this process. And so if we look up, we can see that we have this same exact, uh, chemo trips and active site being regenerated so that it can perform the mechanism all over again on a new substrate. And so really, that is the recap of common traipses catalytic mechanism. And as we move forward in our course will be able to get some practice. So I'll see you guys in our next video.
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Problem

In the mechanism of chymotrypsin, serine-195:

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Problem

Sequentially number the following steps of Chymotrypsin’s catalytic mechanism in the correct order from 1-8:

a) Released newly formed amine portion of the substrate diffuses away _______

b) His-57 deprotonates the Ser-195 hydroxyl group, generating a stronger nucleophile ______

c) Tetrahedral intermediate collapses & His-57 donates a H+ to N of scissile bond (cleaved peptide bond) ______

d) Via general-base-catalysis, His-57 deprotonates a water molecule, generating OH- ______

e) Tetrahedral intermediate collapses & His-57 donates a proton to Ser-195 (cleaving ester bond) _______

f) Released newly formed carboxylic acid portion of the substrate diffuses away & enzyme is restored _______

g) Hydroxide ion attacks the carbonyl group of the substrate, forming another tetrahedral intermediate ______

h) Nucleophilic Ser-195 attacks the carbonyl C of the substrate forming a tetrahedral intermediate______

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