Peptidases - Online Tutor, Practice Problems & Exam Prep

Peptidases, or enzymes that catalyze the hydrolysis of peptide bonds, play crucial roles in protein digestion. Trypsin cleaves specifically after lysine (K) and arginine (R), while chymotrypsin prefers aromatic residues like phenylalanine (F), tyrosine (Y), and tryptophan (W), but can also cleave leucine (L) and methionine (M) slowly. Other peptidases include elastase, thrombin, and pepsin, each with unique cleavage preferences. Notably, proline can inhibit cleavage, affecting all peptidases. Understanding these specificities is essential for grasping protein metabolism and enzymatic functions.

Peptidases

In this video, we're going to begin our discussion on peptidases. So by now, you guys have probably picked up that peptidases are just enzymes, and we know that because it ends in "ase." And anything that ends in "ase" is a good indicator that it's probably an enzyme. Peptidases are indeed enzymes that selectively catalyze the hydrolysis of very specific peptide bonds. And really that's just a fancy way of saying that peptidases are enzymes that cleave or break down very specific peptide bonds. We know this from our previous lessons, where we learned that hydrolysis is just a process of breaking down peptides. Some of these peptidases include trypsin and chymotrypsin, both of which are biologically relevant to our digestive systems. They help us break down proteins found in foods into smaller peptide fragments that our cells can use.

Moving forward, we're first going to focus on trypsin, and then in a different video, we'll focus on chymotrypsin. Trypsin is one of the more specific peptidases because it only cleaves peptide bonds on the carboxyl side of both lysine and arginine amino acid residues. Recall that lysine's one-letter code is K and arginine's one-letter code is R. This specificity is exactly what we mean in our definition of peptidases. So, peptidases don't just cleave random peptide bonds; they cleave very specific peptide bonds next to very specific amino acid residues.

There's something very important you should note before we get to our example. The cleavage of a peptide bond by a peptidase can be blocked or inhibited if a proline amino acid residue is involved in the peptide bond. If a proline residue is involved, it's likely that the peptide bond will not be cleaved because it will be blocked or inhibited from being cleaved. We'll see an example of this below. In our example, we ask where trypsin will do its peptide bond splitting, not in formal terms but as a way to remember easier, comparing the action of trypsin to a knight's sword, specifically after lysine and arginine.

Let's discuss the mnemonic that helps us remember these amino acids: "dragons eat knights riding horses," where K represents lysine and R represents arginine. Lysine's R group resembles a knight's sword, extended and pointy. It has 4 carbon atoms leading to an amino group at the end of its chain. Arginine, on the other hand, has a chain starting with 3 carbon atoms and ends with a triangular nitrogen, resembling a knight's sword. These features are ideal for splitting things, much like a knight's sword splits a watermelon in half. This is how trypsin cleavage is modeled.

In our tetrapeptide example, with four amino acid residues and an N-terminal with a free amino group and a C-terminal with a free carboxylate group, trypsin will cleave after the lysine and arginine residues. However, there’s a proline residue involved in one of the supposed cleavage sites, and this peptide bond will be blocked and inhibited from being cleaved. Only the bond shown in red will actually be cleaved. This results in an alanine-lysine fragment on one side and an arginine-proline fragment on the other.

In our next video, we'll get more practice with trypsin and the cleavage of peptides and proteins. I'll see you guys in that video.

Draw out each of the peptide fragments that would be generated if the peptide is treated with trypsin.

So this practice problem wants us to draw out each of the peptide fragments that would be generated if the peptide is treated with trypsin. And down below, we can see our peptide and we can see that it's being treated with trypsin. And from our previous lesson video, we know that trypsin does its splitting like a knight's sword, after lysine and arginine amino acid residues. And so, recall that lysine's three-letter code is LYS and arginine's three-letter code is ARG. And so, all we need to do is look for the lysines and arginines in our peptide. And notice that there is only one lysine and one arginine in our peptide. And because trypsin models its peptide bond splitting after a knight's sword, it cleaves after lysine and arginine, or on the carboxyl side of lysine and arginine. And so, one of the things that we always need to keep in mind is the fact that proline is capable of inhibiting or blocking the cleavage of a peptide bond if it's involved in that peptide bond that's supposed to be cleaved. And so looking through our peptide, notice that there is one proline residue here and it's involved with the peptide bond here that's supposed to be cleaved. And so what that means is that proline is actually going to block or inhibit the cleavage of this peptide bond and it's not going to be cleaved. So the only peptide bond that will be cleaved is this one shown here. So here we can write cleaved. And essentially what we're going to get are two fragments, our peptides being split into two fragments. This fragment over here on the far left and this fragment right here on the right. And so just to make things a little bit faster, let's write the one-letter codes for the fragments here. So we have Alanine, Phenylalanine, Lysine, Proline, Methionine, Tyrosine, Glycine, and Arginine fragment. And then, that would be this green fragment right here, the same green fragment that we have over here on the left. And then down below we would have the Serine, Tryptophan, Leucine, and Histidine fragment. And so that would be this fragment here. So, we would generate these two fragments upon treating this peptide with trypsin. So this here, on the left on the right here is our answer, and that concludes this practice problem. So I'll see you guys in our next video where we'll cover chymotrypsin.

Peptidases

So now that we've covered trypsin, in this video we're going to focus on chymotrypsin. Chymotrypsin is different from trypsin in that it has a preference for which amino acid residues it recognizes for cleavage. Chymotrypsin prefers breaking aromatic amino acid residues which are Phenylalanine, Tyrosine, and Tryptophan, or, humorously referred to as 'fat young whippersnappers'. You can think of trypsin and chymotrypsin as brothers. Since they are brothers, they both cleave on the carboxyl side of the residues they recognize for cleavage.

Trypsin is the older, more responsible brother, and it's very particular, cleaving like a knight's sword. Trypsin's cleavage is specific, only after lysine and arginine. On the other hand, chymotrypsin is the younger brother who is much more chill and relaxed. Unlike trypsin's precise cleavage, chymotrypsin will slowly cleave other residues over time, beyond the aromatic amino acids, including leucine and methionine residues.

Below, we have a visualization to help you remember how chymotrypsin conducts its cleavage. Chymotrypsin can be humorously thought of as saying, "Come on, trypsin." This relaxed approach is aimed at suggesting trypsin not be so strict only to cleave after lysine and arginine but to also consider other residues over time. The mnemonic "Free your worries like me" helps us remember the amino acid residues chymotrypsin cleaves, where 'F' stands for phenylalanine, 'Y' for tyrosine, 'W' for tryptophan, and 'like me' for leucine and methionine.

The residues that are color-coded in red are those chymotrypsin starts to cleave beyond its primary preferences of aromatic amino acids. Through the mnemonic 'free your worries like me', you'll remember the residues chymotrypsin recognizes for cleavage. In the current example, a tetrapeptide with four amino acid residues, it’s clear only one amino acid residue is recognized for cleavage: Phenylalanine. Hence, it will cleave after phenylalanine, leaving another fragment with alanine and serine.

Moving forward in our practice problems, we will first assume chymotrypsin will cleave its preferred residues unless indicated otherwise. These preferred residues are aromatic amino acids: Phenylalanine, Tyrosine, and Tryptophan. The leucine and methionine will only be cleaved at a very slow rate if enough time is provided. Hopefully, this explanation helps you remember how chymotrypsin performs its cleavage, and you'll be ready to practice in our next video. I'll see you there.

Draw out the resulting peptide fragments that would be generated if the peptide is treated with chymotrypsin.

So, this practice problem wants us to draw out the resulting peptide fragments that would be generated if the peptide is treated with chymotrypsin. And down below, we can see our peptide, and we can see that it's being treated with chymotrypsin. From our previous lesson video, we know that chymotrypsin has a preference for the amino acid residues that it recognizes for cleavage. The mnemonic that helps us memorize the residues that chymotrypsin recognizes for cleavage is just "free your worries like me." Recall that the aromatic amino acids, Phenylalanine, Tyrosine, and Tryptophan, shown in red here, are the ones that chymotrypsin has a preference for cleaving. Leucine and Methionine are the amino acids that chymotrypsin will cleave at a slower rate over a long enough period of time. In our practice problems, we know that unless the practice problem indicates otherwise, we should assume that chymotrypsin is only going to cleave the aromatic amino acids, the ones it has a preference for. Leucine and Methionine here, because there's no indication that these should be cleaved, will not be considered for chymotrypsin's cleavage. Really, what that means is we're going to be looking for these aromatic amino acids: Phenylalanine, Tyrosine, and Tryptophan. The three-letter codes for Phenylalanine are Phe, for Tyrosine are Tyr, and for Tryptophan are Trp. Looking through our residues here, notice what we have is a Phenylalanine, a Tyrosine, and a Tryptophan. Because chymotrypsin is like trypsin's younger brother, they both cleave on the carboxyl side. The carboxyl side is the side that's closest to the C-terminal end, which would be over here on the right. So, it would be all of these peptide bonds shown in red. But, we cannot forget that if a peptide bond includes a Proline amino acid residue, Proline is capable of blocking and inhibiting the cleavage of a peptide bond. Because we have one Proline residue, found here, and it's involved with the peptide bond that's supposed to be cleaved, it's going to block the cleavage of this peptide bond. This peptide bond here is not going to be cleaved. That means only these two peptide bonds in red are going to be cleaved, so we can write "cleaved" above them. Essentially, if these two peptide bonds are cleaved, it will generate three different fragments. It will generate this fragment here on the far left, this one here in the middle, and, of course, the Leucine-Histidine fragment on the right. We can write the one-letter codes for each of these fragments to speed this up. So, if we start with this fragment here, we have Alanine, Lysine, Phenylalanine, Proline, Methionine, and Tyrosine. That would be our green fragment. Our blue fragment here is going to be Glycine, Arginine, Serine, and Tryptophan. That would be our blue fragment. And then, of course, our last fragment here in yellow is going to be Leucine and Histidine, and that would be this yellow fragment. These are the three fragments that would result from treating this peptide here with chymotrypsin. So our answer is this circled part over here on the right. And so that concludes this practice problem, and I'll see you guys in our next video.

Peptidases

So now that we've covered both trypsin and chymotrypsin, in this video, we're going to talk about some other relevant peptidases that your professor may or may not expect you to memorize. On the chart below, we have a list of these relevant peptidases and the specific peptide bonds that they hydrolyze or the specific peptide bonds that they break or cleave. Something important from our previous lesson videos is the fact that peptidase bond cleavage can be inhibited if a proline amino acid residue participates in the peptide bond that's supposed to be cleaved. This applies to all the peptidases listed in our chart below, so keep that in mind going forward.

In our example below, we've got a chart with three different columns. In the far left column, we have the name of the peptidase. In the middle column, we indicate whether the peptide bond that's cleaved is on the N-terminal side or the C-terminal side of the residue. On the far right column, we have the amino acid cleavage site or the exact amino acid that the peptidase recognizes for cleavage. All of these peptidases are color-coded yellow, indicating they cleave peptide bonds on the C-terminal side of the residue they recognize for cleavage. In this column here, we can note the C-terminal side for all four peptidases.

Specifically for trypsin, as covered in our previous videos, trypsin cleaves after lysine and arginine amino acid residues. Chymotrypsin, akin to trypsin's younger brother, also cleaves on the C-terminal side of the peptide bond. However, it prefers aromatic amino acid residues like Phenylalanine, Tyrosine, and Tryptophan but will also very slowly cleave other residues including Leucine and Methionine. Remember the mnemonic "free your worries like me" to help memorize the residues that chymotrypsin recognizes for cleavage.

The rest of the peptidases listed below have not yet been discussed extensively, so confirming with your professor if these are to be memorized might be wise. Elastase cleaves the C-terminal peptide bonds and prefers amino acid residues with small neutral R-groups, mostly including hydrophobic amino acids except methionine and proline. Serine is also recognized by elastase. Thrombin cleaves peptide bonds on the C-terminal side, but recognizes only arginine amino acid residues, unlike trypsin which also recognizes lysine.

Pepsin is color-coded in blue because it cleaves peptide bonds on the N-terminal side of the residue it recognizes for cleavage. Pepsin recognizes the same amino acids as chymotrypsin, except it does not cleave methionine. Carboxypeptidase A is noted separately because it cleaves off C-terminal residues and specifically, it cleaves the N-terminal peptide bond to do so, excluding arginine, lysine, and proline. We'll get to practice using these peptidases in forthcoming practice problems. Looking forward to seeing you in those videos!

What would the resulting peptide fragments be if the following peptide were treated with excess Pepsin? 

                                H—E—L—P—M—E—P—L—E—A—S—E

• A. H—E—L—P—M—E—P—L & E—A—S—E
• B. H—E—L—P, M—E—P, & L—E—A—S—E
• C. H—E, L—P—M—E—P—L—E—A—S—E
• D. H—E, L—P—M—E, P—L—E, A—S—E

Which is the expected result of chymotrypsin cleavage of the following peptide? 

                            Lys—Gly—Phe—Thr—Tyr—Pro—Asn—Trp—Ser—Tyr—Phe

• A. Lys—Gly—Phe, Thr—Tyr—Pro—Asn—Trp, Ser—Tyr, & Phe
• B. Lys—Gly—Phe, & Thr—Tyr—Pro—Asn—Trp—Ser—Tyr—Phe
• C. Lys—Gly—Phe, Thr—Tyr—Pro—Asn—Trp, Ser, & Tyr—Phe
• D. Lys—Gly—Phe—Thr—Tyr—Pro—Asn—Trp, Ser, & Tyr—Phe

You perform multiple tests to derive the amino acid sequence of a purified peptide (see results below). Which of the following peptides listed best represents the sequence of the unknown peptide?

• A. Trp—Tyr—Ala—Ala—His.
• B. Ala—His—Trp—Tyr—Ala.
• C. His—Ala—Trp—Tyr—Ala.
• D. Ala—His—Tyr—Trp—His.
• E. His—Ala—Tyr—Trp—Ala.

A) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Would ion exchange or size-exclusion chromatography be most appropriate for separating the fragments? Explain. 

a. Ion-Exchange chromatography.

b. Size-exclusion chromatography.

B) Suppose the same peptide was digested with chymotrypsin. Which would be the optimal separation technique? Explain.

a. Ion-Exchange chromatography.

b. Size-exclusion chromatography.

So this practice problem is broken up into 2 different parts. Part a, up above, and part b, down below. Part a up above says the octapeptide, or a peptide with 8 amino acid residues in it, shown here, was digested with the enzyme trypsin. And then it asks, would ion exchange or size exclusion chromatography be most appropriate for separating the fragments? Explain. And the answer options have either ion exchange or size exclusion chromatography. And so down below here, notice I've redrawn the same exact peptide that we have up above. And we know that we're going to be treating it with trypsin and from our previous lesson videos, we know that trypsin does its cleaving after a knight's sword. And so it cleaves after lysine and arginine residues on the carboxyl side. And so looking at our peptide here, notice that there is an arginine here and a lysine here. And so it's going to cleave the carboxyl side of these two residues on the peptide bond on the carboxyl side. So that means it's going to cleave right here, as well as right over here. And notice that there are no prolines in our residue, so we don't have to worry about the inhibition or blockage of a proline residue. And so this cleavage is going to result in 3 different fragments. This fragment over here on the far left, this fragment here in the middle, and the serine free amino acid residue on its own. And so essentially, what we can do is determine the charges of each of these fragments, since we know that ion exchange chromatography will separate fragments based on their charges. And so, with this first fragment here, we can redraw it over here, so AVGWR. And, we know that there are only 7 amino acids with ionizable R groups and the mnemonic to help us memorize those 7 amino acid ionizable R groups is just yucky crazy dragons eat knights riding horses. And so, what we'll see is that none of these amino acids have ionizable R groups except for arginine. And so essentially what we can do is consider the charge of arginine. And so, we know that at a pH of neutral pH, that arginine is going to have a positive charge. So we'll consider everything around neutral pH. Now, none of these other amino acids here have ionizable R groups. So now all we need to worry about is the ionizable amino group, which we know is going to have a positive charge at neutral pH, and the ionizable carboxyl group, which we know is going to have a negative charge at neutral pH. And so, the total charge for this green fragment here is going to be plus one. That's for this green fragment. Now, if we do the same for this blue fragment here, we have valine and lysine. And lysine, notice, is one of the amino acids with an ionizable R group. And at neutral pH, it has a potential, it has a positive, charge. So we can put a positive charge here. And valine does not have an ionizable R group because it's not one of these amino acids up here. So all we need to do is consider the amino group, which we know is going to have a positive charge, and the carboxyl group, which we know is going to have a negative charge at neutral pH. And so the total charge for this fragment is going to be the sum of these charges, so it's just going to be plus 1. And then of course, the serine at the very end here is going to be all a free amino acid on its own. It does not have an ionizable R group, so it's just going to be the amino group as well as the carboxyl group. And so the amino group is going to have a positive charge at neutral pH, and the carboxyl group will have a negative charge at neutral pH. So this is going to have an overall neutral net charge of 0. And so essentially what we'll see here is that this light blue fragment and this green fragment have the same charges. So using ion exchange chromatography is not going to be a good technique to separate them. If the fragments are different sizes, then size exclusion chromatography can be a good technique to separate the 3 fragments. Notice we only have one residue here. The light blue fragment is double the size, or nearly double the size of the serine fragment. And then this green fragment has 5 amino acid residues in it, so it's much larger than the other 2. So, separating based on size is going to be the best way. So, that means for part a, answer option b is the correct choice. So, we can mark b as being correct. And the other option is incorrect, so we can scratch it off. And so, essentially, the explanation for why is again because the charges of this fragment here, as well as this fragment are identical. So, ion exchange chromatography would not be good to separate these two fragments. Now, moving on to our next part down below, part b, notice that it says that, suppose the same exact exact peptide from above is digested, but this time with chymotrypsin. Which would be the optimal separation technique, ion exchange or size exclusion chromatography? Explain. So recall that chymotrypsin has a preference for cleaving aromatic amino acid residues, and the mnemonic to help us memorize that is: Free your worries like me. And so remember that the aromatic amino acids, phenylalanine, tyrosine, and tryptophan are the ones that it recognizes and has a preference for cleaving. And leucine and methionine here are residues that it cleaves at a much slower rate over a longer period of time. And since there's no indication that Leucine and Methionine are actually going to be cleaved here, we are only going to assume that chymotrypsin is going to cleave the aromatic amino acid residues here. And since chymotrypsin is like trypsin's younger brother, they both cleave the C-terminal peptide bond. And so looking at our peptide, notice that there's only one aromatic amino acid residue and that's tryptophan, and it's going to cleave the C-terminal peptide bond shown here. And so essentially what's going to happen is we're going to break these 2 break our fragment in half. So we'll have this peptide fragment on the left and this peptide fragment on the right. And so essentially what you'll see is that they both have 4 amino acid residues in them. So what that means is that they're going to have approximately the same size and size exclusion chromatography is probably not going to be the best idea to separate these two fragments. So, let's draw out these fragments here separated. And essentially what we can do just to check is we can check to see what the charges are going to be for these 2 separate fragments. So, we already anticipate that size exclusion chromatography is not going to be a good idea since, again, they have approximately the same size, since they have the same number of amino acid residues. But, again, the mnemonic to help us memorize the 7 amino acids with ionizable R groups is just yucky crazy dragons eat knights riding horses. And so if it's not one of these 7 amino acids, then its R group is not going to be ionizable. So what you'll see is that for this first group over here, none of the amino acids have ionizable R groups. So, we're only going to have an ionizable amino group and an ionizable carboxyl group at pH 7. And it's going to be positive for the amino group and negative for the carboxyl group, meaning that the overall charge for this green fragment is going to be 0. Now, for the blue fragment, notice that we have 2 groups that have ionizable R groups, and that is arginine and lysine, which both fall up here, arginine and lysine. And so at physiological pH, right around the pH of 7, they both have positively charged R groups, so we can put a positive sign in here. And then for the amino group, we know that at pH of 7, it's going to have a positive charge, and the C-terminal end over here is going to have a negative charge. And so essentially the overall charge for this fragment is going to be plus 2, since it's the sum of these 4 charges here. And so, a plus 2 charge is much different than a 0 charge. And so since these two charges differ by so much, ion exchange chromatography is probably the better technique to separate them. So that means that answer a here is actually the correct answer. Now, recall from our previous lessons that there are 2 different types of ion exchange chromatography, cation exchange and anion exchange chromatography. So, which do you think would be better for separating these two fragments? Cation exchange or anion exchange? Well, it's actually cation exchange chromatography. And the reason is because, remember with a positively charged fragment like this, it's going to remain stuck inside of the column longer. It's going to have more interactions with the mobile phase and the stationary phase, which means it's going to get better separation. And so cation exchange chromatography would be best. And so that concludes this 2 part practice problem, and I'll see you guys in our next video.

A nonapeptide was determined to have the following amino acid composition: (Lys)₂, (Gly)₂, (Phe)₂, His, Thr, Met.  The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB) and then hydrolyzed; 2,4-dinitrophenylhistidine was identified by HPLC.  When the native peptide was exposed to cyanogen bromide (CNBr), an octapeptide and free glycine were recovered.  Incubation of the native peptide with trypsin gave a pentapeptide, a tripeptide, and free Lys.  2,4-Dinitrophenyl-histidine was recovered from the pentapeptide, and 2,4-dinitrophenylphenylalanine was recovered from the tripeptide.  Digestion with the enzyme pepsin produced a dipeptide, a tripeptide, and a tetrapeptide.  The tetrapeptide was composed of (Lys)₂, Phe, and Gly.  The native sequence was determined to be:

*Recall: Pepsin cleaves N-terminal peptide bond of F, Y, W & L residues.*

• A. Gly—Phe—Lys—Lys—Gly—Thr—Met—Phe—His.
• B. His—Thr—Gly—Lys—Lys—Phe—Phe—Gly—Met.
• C. His—Thr—Phe—Gly—Lys—Lys—Phe—Met—Gly.
• D. His—Phe—Thr—Gly—Lys—Lys—Phe—Met—Gly.
• E. Met—Thr—Phe—Lys—Phe—Gly—Gly—Lys—His.

The following reagents are often used in protein chemistry. Match the reagent with the purpose for which it is best suited. Some answers may be used more than once or not at all and more than one reagent may be suitable for a given purpose.

Hey, guys. So, this practice problem just wants us to do some matching. And it says the following reagents are often used in protein chemistry, and we've got the reagents listed on the far left. And then it says, match the reagent with the purpose for which it is best suited. Some answers may be used more than once or not at all, and more than one reagent may be suitable for a given purpose. And so, the first purpose that we have here is the hydrolysis of peptide bonds on the C-terminal side of lysine and arginine. Lysine and arginine's one-letter codes are K and R. And, of course, we know that trypsin is the enzyme, the peptidase, that will cleave the peptide bonds on the C-terminal side of lysine and arginine, and that's because trypsin does its splitting like a knight's sword after lysine and arginine. And so, we can go ahead and put in F here for trypsin, and we can move on to our next purpose. So the next purpose says, cleavage of peptide bonds on the C-terminal side of methionine. And this, of course, is going to be cyanogen bromide. So we know that if we take the B here in this abbreviation and we rotate it 90 degrees counterclockwise, it literally tells us where it cleaves, and it cleaves next to Methionine residues. And so, it cleaves the C-terminal side because cyanogen bromide starts with a C. And so, what that means is for this purpose here, we can put answer option A. Alright. So, moving on to the next purpose, what we have is breakage of disulfide bonds. And it turns out that there's actually multiple chemicals that can be used to break disulfide bonds. We know that beta mercaptoethanol can be used to break disulfide bonds, but also recall from our diagonal electrophoresis lesson that performic acid can also be used to cleave disulfide bonds. And so what that means is we can put in both option D and option H for this purpose. Now, moving on to our next purpose, it says carboxymethylation of cysteines to prevent disulfide reformation. And of course, this is referring to iodoacetate. So iodoacetate is used in conjunction with beta mercaptoethanol. Beta mercaptoethanol reduces the disulfide bonds to break them into cysteinesulf hydro groups. And then iodoacetate is used to carboxymethylate those cysteines to prevent the reformation of the disulfide bonds. And that's because we want to permanently break the disulfide bonds since disulfide bonds can interfere with the sequencing procedure. And so, what this means is that we can put G here for this purpose. Now, moving on to the next purpose; it says determining the N-terminal amino acid residue in a polypeptide. And it turns out that FDNB is one of those molecules that reacts to covalently label the N-terminal amino acid residue. And ultimately, it can help reveal the N-terminal amino acid residue of a polypeptide. So we can put in answer option C here. And, moving on to our next purpose, what we have is determining the C-terminal amino acid residue in a polypeptide. And this, of course, is going to be hydrazine. So the Z, just like Z is the last letter of the alphabet, hydrazine is used to identify the last residue of a polypeptide chain, or the C-terminal residue. And so, what that means is that we can put for this purpose here, hydrazine, which is letter I. And, moving on to our next purpose, essentially, what we have is the determination of the amino acid sequence of a peptide. And so, we have not yet talked about Edman degradation sequencing, but that is our next lesson video. And it turns out that this chemical here, which is the only chemical on this list that we haven't yet talked about, is used for Edman degradation sequencing and determining the amino acid sequence of a peptide. And so, what that means is we can put in letter J here as well. Now, it turns out, that the way that sequencing works, that this component here can actually be used to determine the N-terminal amino acid residue in a polypeptide as well. So, you guys will be able to understand that a lot better when we get to our next lesson video which is going to talk about the overview of Edman degradation sequencing. So, this here concludes our practice problem, and I'll see you guys in our next video.

Here’s what students ask on this topic:

What are peptidases and what role do they play in protein digestion?

Peptidases are enzymes that catalyze the hydrolysis of peptide bonds in proteins. They play a crucial role in protein digestion by breaking down proteins into smaller peptide fragments and amino acids, which can be absorbed and utilized by the body. Specific peptidases, such as trypsin and chymotrypsin, target particular peptide bonds. For instance, trypsin cleaves after lysine (K) and arginine (R), while chymotrypsin prefers aromatic residues like phenylalanine (F), tyrosine (Y), and tryptophan (W). Understanding the specificity of these enzymes is essential for comprehending protein metabolism and enzymatic functions in biological systems.

How does trypsin specifically cleave peptide bonds?

Trypsin is a highly specific peptidase that cleaves peptide bonds on the carboxyl side of lysine (K) and arginine (R) residues. This specificity is due to the enzyme's active site, which is structured to recognize and bind to these amino acids. The cleavage occurs through a hydrolysis reaction, where a water molecule is used to break the peptide bond. However, if a proline residue is involved in the peptide bond, the cleavage is typically inhibited. This specificity ensures that trypsin selectively breaks down proteins into smaller, manageable fragments for further digestion and absorption.

What is the difference between trypsin and chymotrypsin in terms of their cleavage preferences?

Trypsin and chymotrypsin are both peptidases but have different cleavage preferences. Trypsin specifically cleaves peptide bonds on the carboxyl side of lysine (K) and arginine (R) residues. In contrast, chymotrypsin prefers aromatic amino acids such as phenylalanine (F), tyrosine (Y), and tryptophan (W). Additionally, chymotrypsin can slowly cleave leucine (L) and methionine (M) residues over time. This difference in specificity allows these enzymes to work together in the digestive system to break down a wide variety of protein substrates efficiently.

Why is proline known to inhibit the cleavage of peptide bonds by peptidases?

Proline inhibits the cleavage of peptide bonds by peptidases due to its unique cyclic structure, which imposes conformational constraints on the peptide chain. This rigidity makes it difficult for the enzyme to access and hydrolyze the peptide bond involving proline. As a result, when a proline residue is part of the peptide bond, it often blocks or inhibits the cleavage by peptidases such as trypsin, chymotrypsin, and others. This inhibition is a crucial consideration in understanding the specificity and function of peptidases in protein digestion.

What are some other relevant peptidases besides trypsin and chymotrypsin, and what are their specific cleavage preferences?

Besides trypsin and chymotrypsin, other relevant peptidases include elastase, thrombin, pepsin, and carboxypeptidase A. Elastase cleaves peptide bonds on the C-terminal side of small, neutral amino acids. Thrombin specifically cleaves after arginine (R) residues. Pepsin cleaves on the N-terminal side of aromatic residues like phenylalanine (F), tyrosine (Y), and tryptophan (W), as well as leucine (L). Carboxypeptidase A cleaves off C-terminal residues, except for arginine (R), lysine (K), and proline (P). Understanding these specificities helps in studying protein metabolism and enzymatic functions.