Chymotrypsin's Catalytic Mechanism - Online Tutor, Practice Problems & Exam Prep

Chymotrypsin's catalytic mechanism consists of two phases: acylation and deacylation. In the acylation phase, substrate binding occurs, followed by a nucleophilic attack by serine 195, forming a tetrahedral intermediate stabilized by the oxyanion hole. This leads to the cleavage of the peptide bond and the formation of an acyl enzyme via a covalent ester linkage. The deacylation phase involves water acting as a nucleophile, regenerating the original enzyme by hydrolyzing the ester bond. This mechanism highlights the importance of covalent catalysis and enzyme-substrate interactions in biochemical reactions.

Chymotrypsin's Catalytic Mechanism

In this video, we're going to begin our discussion on chymotrypsin's catalytic mechanism. Chymotrypsin's catalytic mechanism can actually be broken up into 2 separate phases. The first phase is referred to as the acylation phase, and the second phase is referred to as the deacylation phase. Notice that we have both of these phases color coded below, where the yellow background represents the acylation phase and the blue background represents the deacylation phase. Moving forward in our course, we will talk about each of these two phases in a lot more detail.

Now, notice that in the acylation phase, we're starting off with a yellow half-circle that represents chymotrypsin's active site, which we know has the catalytic triad. However, we're only showing the serine amino acid residue of the catalytic triad in this particular image. We know that the catalytic triad will establish the serine as a stronger nucleophile, which is why it has this negative charge on it. Chymotrypsin is all about cleaving specific peptide bonds, more specifically, the C-terminal peptide bonds of aromatic amino acid residues. Here in pink, we have a peptide bond that is going to be cleaved by chymotrypsin.

In the first phase, the acylation phase, part of our substrate is going to be cleaved off covalently. In the acylation phase, the peptide bond of interest that we were trying to cleave has already been cleaved. However, the acylation phase has acylated our enzyme, resulting in an acyl enzyme that is covalently modified. In the deacylation phase, it's all about regenerating the original enzyme so that the mechanism can occur again. Notice that water will come into play here to hydrolyze this bond and release this portion of the substrate and regenerate our original enzyme. This hydrogen can be removed through the catalytic triad to establish serine once again as a strong nucleophile as it was before.

We'll be able to talk more about each of these two phases as we move forward in our course. In our next video, we'll discuss the details about the acylation phase. I'll see you guys in that video.

Chymotrypsin's Catalytic Mechanism

In this video, we're going to talk about chymotrypsin's first phase of its catalytic mechanism, which is the acylation phase. Chymotrypsin's acylation phase can really be broken down into 4 general steps that we'll talk about below. The main takeaway of chymotrypsin's 4-step acylation phase is the formation of covalent catalysis that forms an ester linkage, covalently linking chymotrypsin's active site with the substrate, as we'll see below. However, this covalent ester linkage, formed in the acylation phase, is just a temporary covalent linkage because, in the second phase, the deacylation phase that we'll talk about in our next video, this covalent linkage will be broken. In addition to this ester linkage that is created in the acylation phase, the peptide bond of interest that chymotrypsin is supposed to cleave is actually going to be broken or cleaved. The acylation phase is really just a nucleophilic and is a catalytic mechanism that you probably covered in your previous organic chemistry courses.

Again, the acylation phase can be broken down into these 4 general steps: substrate binding, nucleophilic attack, removal of the leaving group or the LG for short, and end of the phase. Notice that in our image, the numbers correspond with the ones we have described. Starting with the first phase, substrate binding, speaks for itself. The substrate of chymotrypsin is the peptide that's going to be cleaved and will bind to chymotrypsin's active site. If we look at our image over here on the far left-hand side, we have chymotrypsin's active site, which has the catalytic triad we discussed in our previous lesson videos, which are aspartate 102, histidine 57, and serine 195, and these three amino acids will be seen throughout the entire catalytic mechanism.

Notice that the substrate is a peptide with an amino end with NH3+, and a carboxyl end with a carboxylate group. We have four total amino acids represented by circles: Alanine and Valine, which are non-polar but not aromatic, and Arginine, a charged amino acid but not aromatic. The only aromatic amino acid here is Phenylalanine, recognized by chymotrypsin for cleavage and shown in a darker shade of purple. Chymotrypsin will cleave the peptide bond closest to the c-terminal end, shown in pink.

In the first step of nucleophilic attack, since serine 195 has been established as a strong nucleophile, it will perform a nucleophilic attack on the carbonyl carbon of the substrate to form an unstable tetrahedral intermediate. Histidine 57 acts as a base, acquiring the hydrogen on Serine, allowing the electron density to attack the carbonyl carbon atom of the substrate. This forms a tetrahedral intermediate that is unstable, yet stabilized by the oxyanion hole on chymotrypsin's active site. This covalent catalysis forms a covalent ester linkage between chymotrypsin's serine 195 and the carbonyl carbon of the substrate.

The third step involves the removal of the leaving group, LG for short. The tetrahedral intermediate collapses; Histidine 57 acts as an acid and the peptide bond is cleaved. This reaction arrow represents the collapse of the tetrahedral intermediate, with the electron density in the peptide bond being used to perform a nucleophilic attack on this acidic hydrogen, resulting in the bond being cleaved. Notice that the peptide bond is no longer there, as it was above, and histidine ends up as the conjugate base without a charge.

The fourth step marks the end of the acylation phase. An enzyme acylated and the released amine portion of the substrate will freely diffuse away. This concludes the formation of the acyl enzyme, setting the stage for the deacylation phase, where this portion of the substrate will be removed and the original enzyme restored. The main takeaway of chymotrypsin's acylation phase is that covalent catalysis forms an ester linkage that creates the acyl enzyme, and the peptide bond of interest is cleaved. That concludes this video, and I'll see you in our next video where we'll discuss the deacylation phase.

Chymotrypsin's Catalytic Mechanism

So now that we've covered the first phase of chymotrypsin's catalytic mechanism, in this video we're going to move on to the second phase of chymotrypsin's catalytic mechanism, which is the deacylation phase. Now, just like the acylation phase, chymotrypsin's deacylation phase can be summed up in 4 general steps that we'll talk about down below. Now, because the deacylation phase is really just a continuation of the acylation phase, we've continued the numbering system down below starting with number 5. Now, really, the main takeaway of chymotrypsin's 4 step deacylation phase is right here, and that is that the covalent ester linkage that was formed in the acylation phase is going to be hydrolyzed or broken in order to regenerate or restore the original chymotrypsin enzyme so that it can again perform the same exact catalytic mechanism once again on a different substrate. And so what we'll notice is that this deacylation phase of chymotrypsin's catalytic mechanism is really just an ester hydrolysis, 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 acylation phase, so we still have substrate binding, nucleophilic attack, removal of the 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 the 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 deacylation phase, there's going to be a new substrate that's going to enter the chymotrypsin active site, and that is going to be a water molecule. An H₂O molecule is going to come into play. And, once this H₂O, water molecule enters chymotrypsin's active site, the remainder of the mechanism is gonna be pretty much a series of very similar steps, that are gonna repeat from the acylation phase. So, what we'll see is down below, we have the acyl enzyme that, we left off with in the acylation phase. And, what you'll notice is that in the very, the 5th step here, which is, the first step of the deacylation phase, a water molecule, an H₂O molecule, is entering into the active site. And so in the 6th, step here, the nucleophilic attack, histidine 57 is going to act as a base to deprotonate that water molecule, and that's going to create a hydroxide ion that is going to act as a nucleophile and attack the carbonyl carbon atom of this portion of the substrate here, and that is going to once again create a tetrahedral intermediate. And so if we take a look at our image down below, we can see the water molecule comes into place. Here's the water molecule. And then histidine 57 here is going to act as a base through general base catalysis, 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 carbonyl carbon atom of this portion of the substrate here. And so this is going to generate a tetrahedral intermediate once again. And so, this tetrahedral intermediate here, is going to be unstable. However, once again, we can say that the oxyanion hole, which is again a region in chymotrypsin's active site, is going to stabilize the tetrahedral intermediate in order to ensure that it can readily form. And so in the 7th step here, what we have is removal of the leaving group. And so in order for that to happen, the tetrahedral intermediate is going to collapse once again. Histidine 57 is going to act as an acid again, and the ester bond is going to be broken. And so if we take a look down below, notice that in the 7th step here, what happens is this tetrahedral intermediate is going to collapse through this arrow right here. And then this, histidine 57 is going to act as an acid and donate this blue hydrogen here. And, of course, the electron density here forming the ester bond is going to pick up that hydrogen, and, that is going to cause this ester linkage here to be broken. And so that's exactly what we're showing here in the 7th step is that the ester bond is being cleaved. And so notice that we have the ester, bond up above here. However, over here, it has been cleaved and it's no longer present. And so, in the 8th and final step, we have the end of the phase. And so at this point, the enzyme, chymotrypsin, has been deacetylated, and the released carboxylic acid portion of the substrate is free to diffuse away. And so we can see here, we have this released carboxylic acid portion and it is free to diffuse away. And, ultimately, what we're left with is the original chymotrypsin enzyme, which is now restored back to its original state, and it's now ready for another round of catalysis. And so, really, that is all of the chymotrypsin catalytic mechanism. It's broken up into the acylation phase and the deacylation phase, And the steps are very similar and, very parallel. And so in our next video, we'll be able to, put together both the acylation and deacylation phase, into one video so that we can look at, the entire catalytic mechanism all at once. So, that concludes this video and I'll see you guys in our next one.

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

• A. General-Base catalysis; 1 ; covalent catalysis; 2.
• B. General-Acid catalysis; 1 ; covalent catalysis; 3.
• C. Covalent catalysis; 2 ; covalent catalysis; 3.
• D. Covalent catalysis; 1 ; General-Base catalysis; 3.

Chymotrypsin's Catalytic Mechanism

In this video, we're going to do a recap of chymotrypsin's entire catalytic mechanism, including both the acylation and deacylation 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 interactively 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 acylation phase. And then the steps that have a blue background are correlating with the deacylation phase. And so, what you'll notice is over here in the far top left, we're showing you chymotrypsin's catalytic mechanism, which of course has the catalytic triad, which are the 3 amino acids, aspartate 102, histidine 57, and serine 195. And so in the first stage, the first step of the acylation phase, we have binding of the substrate. So here is our substrate, and it's going to be bound to chymotrypsin's active site. Now in the second step, what we're going to have is a nucleophilic attack. And so recall that histidine 57 here is going to be hydrogen binding with serine 195 and aspartate 102, all to make histidine 57 act as a better base so that it can make serine 195 a better nucleophile. And so, essentially, what's going to happen is histidine 57 here is going to act as a base and take up this hydrogen right here, And then the extra electron density right here, is going to attack a nucleophilic attack on this carbonyl carbon atom. And then, of course, the electron 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 covalent catalysis because a new covalent bond is forming between the enzyme's active site as well, with the substrate. And so in the second step, what we end up generating is a tetrahedral intermediate, which is what we have here. And recall that the tetrahedral intermediate is going to be unstable and so, a portion of chymotrypsin's active site referred to as the oxyanion hole is going to help stabilize the tetrahedral intermediate that we have here. And of course, the third step is going to be the removal of the leaving group. And so in this third step, essentially, what's going to happen is the tetrahedral intermediate, up here is going to collapse. Histidine 57 is going to act as an acid, and so it's going to essentially donate this hydrogen right here. And, let's make this arrow a little clearer. And, this hydrogen's going to be donated, and it's going to be collected and picked up by the peptide bond here that's going to ultimately be broken. And so in the 3rd step, we do have 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 diffuse away, and we're left with a covalent attachment here between the substrate and the enzyme. And so this is all referred to as the acyl enzyme. And this is exactly where we pick up when we start the deacylation phase. And so notice that with the deacylation phase, again, we're going to repeat the similar steps. So we're going to have the introduction of a substrate. And so now we're introducing a new substrate, water. So a water molecule is going to come into the active site. And so we're going to have a series of similar steps. And, in step 6, essentially what's going to happen is a nucleophilic attack. And so histidine 57 is again going to help establish a strong nucleophile. So it is going to act as a base and collect a hydrogen atom from the water molecule. And then this extra electron density here on the water molecule, which would be generating a hydroxide ion, would be able to attack perform a nucleophilic attack on the carbonyl carbon atom again. And, of course, the electron density here in the double bond is going to have to shift up. And so ultimately this generates a tetrahedral intermediate that is relatively unstable but is stabilized through the oxyanion hole of chymotrypsin's active site. And so, in the next step what we have is the removal of the leaving group and the leaving group here is actually going to be the remainder of the substrate. It's going to 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 tetrahedral collapse, and then the electron density here in this ester bond is going to be used to perform a nucleophilic 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 this, this ester bond is being cleaved, and so that's exactly why we have ester bond being cleaved here, and you can see that up above the ester 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 diffuse away and active site that active site that we had at the very beginning of this process. And so if we look up, we can see that we have the same exact chymotrypsin 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 chymotrypsin's catalytic mechanism. And as we move forward in our course, we'll be able to get some practice. So I'll see you guys in our next video.

In the mechanism of chymotrypsin, serine-195:

• A. Functions as a nucleophile.
• B. Functions as an electrophile.
• C. Forms a tetrahedral intermediate with part of the substrate.
• D. Is reduced by Aspartate 102.
• E. Is oxidized by Histidine 57.
• F. A and C.
• G. A, C and E.

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______

So this practice problem wants us to sequentially number the following steps of chymotrypsin's catalytic mechanism in the correct order from 1 to 8, and we've got these 8 steps listed down below. What we need to recall from our previous lesson videos is that chymotrypsin's catalytic mechanism is broken up into 2 phases, the acylation phase and the deacylation phase. These steps can be broken into similar categories. Notice we have the acylation phase over here on the left, which has substrate binding, nucleophilic attack, removal of the leaving group, and the end of the phase. On the right, we have the deacylation phase, which also consists of substrate binding, nucleophilic attack, removal of the leaving group, and the end of the phase. By remembering these general classes of steps, we can correctly order the steps of chymotrypsin's catalytic mechanism. After reviewing each of these steps, the first step is going to be option b. We can number it number 1 because it says Histidine57deprotonatestheSerine195hydroxylgroupgeneratingastrongernucleophile. This step occurs in chymotrypsin's active site even before the acylation phase begins, functioning as step 0 technically, but for this, here it is the first step. The next step, after the stronger nucleophile has been generated, is for Serine195 to perform a nucleophilic attack on the carbonyl carbon of the substrate forming a tetrahedral intermediate. This is really step 2 here, the nucleophilic attack of the acylation phase and corresponds with option h. The collapse of the tetrahedral intermediate generated in step 2, where Histidine57 donates its acidic hydrogen to the nitrogen of the peptide bond that is cleaved, is designated as step 3 and corresponds with option c. The end of the acylation phase, where the newly formed amine portion of the substrate diffuses away, marks step 4 and corresponds with option a. Transitioning to the deacylation phase, which begins with the entry of a water molecule into the active site, step 5 corresponds with option d, where Histidine57 deprotonates the water molecule, generating a hydroxide ion. The nucleophilic attack by the hydroxide ion on the carbonyl group of the substrate forms another tetrahedral intermediate. This is represented as step 6 and corresponds with the nucleophilic attack in the deacylation phase as described. The removal of the leaving group in the deacylation phase, step 7, sees the collapse of the tetrahedral intermediate formed in step 6, as described in option e, where Histidine57 donates its acidic proton to Serine195 to cleave the ester bond. The final step, step 8, is the end of the deacylation phase, where the newly formed carboxylic acid portion of the substrate diffuses away, restoring the chymotrypsin enzyme back to its original state to begin the acylation phase again. This concludes our practice problem and the best way to test yourself and see if you really understand chymotrypsin's catalytic mechanism is to revisit this practice problem and try to order these steps correctly.

Here’s what students ask on this topic:

What are the two phases of chymotrypsin's catalytic mechanism?

Chymotrypsin's catalytic mechanism consists of two phases: the acylation phase and the deacylation phase. In the acylation phase, the substrate binds to the enzyme's active site, and serine 195 performs a nucleophilic attack on the carbonyl carbon of the substrate, forming a tetrahedral intermediate. This intermediate is stabilized by the oxyanion hole, leading to the cleavage of the peptide bond and the formation of an acyl-enzyme complex via a covalent ester linkage. In the deacylation phase, a water molecule acts as a nucleophile, hydrolyzing the ester bond to regenerate the original enzyme, allowing it to catalyze another reaction.

How does the catalytic triad in chymotrypsin function?

The catalytic triad in chymotrypsin consists of three amino acids: aspartate 102, histidine 57, and serine 195. These residues work together to enhance the nucleophilicity of serine 195. Aspartate 102 stabilizes histidine 57 through hydrogen bonding, making histidine a better base. Histidine 57 then deprotonates serine 195, converting it into a strong nucleophile. This nucleophilic serine 195 can then attack the carbonyl carbon of the substrate, forming a tetrahedral intermediate and facilitating peptide bond cleavage.

What role does the oxyanion hole play in chymotrypsin's catalytic mechanism?

The oxyanion hole in chymotrypsin's active site plays a crucial role in stabilizing the tetrahedral intermediate formed during the catalytic process. When serine 195 attacks the carbonyl carbon of the substrate, the resulting tetrahedral intermediate is unstable. The oxyanion hole, a region in the active site, stabilizes this intermediate by forming hydrogen bonds with the negatively charged oxygen atom. This stabilization is essential for the intermediate to form and proceed to the next steps of the catalytic mechanism, ultimately leading to peptide bond cleavage.

What is the significance of covalent catalysis in chymotrypsin's mechanism?

Covalent catalysis is a key feature of chymotrypsin's catalytic mechanism. During the acylation phase, serine 195 forms a covalent ester linkage with the substrate, creating an acyl-enzyme intermediate. This covalent bond is crucial for the cleavage of the peptide bond. In the deacylation phase, water acts as a nucleophile to hydrolyze this ester bond, regenerating the original enzyme. Covalent catalysis allows for a more controlled and efficient cleavage of peptide bonds, highlighting the enzyme's specificity and catalytic power.

How does water contribute to the deacylation phase in chymotrypsin's mechanism?

In the deacylation phase of chymotrypsin's catalytic mechanism, water acts as a nucleophile. A water molecule enters the active site and is deprotonated by histidine 57, forming a hydroxide ion. This hydroxide ion then performs a nucleophilic attack on the carbonyl carbon of the acyl-enzyme intermediate, forming a tetrahedral intermediate. The oxyanion hole stabilizes this intermediate, allowing it to collapse and break the ester bond. This hydrolysis reaction regenerates the original enzyme, making it ready for another catalytic cycle.