Serine Proteases – Enzyme Catalysis

by Kevin Ahern, PhD

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    00:00 Okay. Now, serine proteases, as I said, cleave peptide bonds, that's the catalytic thing that they do.

    00:06 They have specificity of cutting, again, by binding only to certain proteins.

    00:12 They only cut those proteins that they bind. They have a common active site.

    00:18 All the serine proteases, the different serine proteases have a three dimensional configuration of the place in them where the reaction occurs.

    00:27 Now we will see that that is important because that configuration is what creates the electronic environment necessary for the reaction to take place.

    00:37 And last of all, the serine proteases are very well studied. So we understand the mechanism of their action quite well.

    00:43 So let's take a look now at the mechanism of the serine proteases.

    00:47 I have shown on the screen here a substrate for the enzyme. This is a polypeptide chain or protein that the serine protease will cut.

    00:58 The specific cut is going to occur here will occur between the carbon and the nitrogen on this molecule.

    01:04 And of course, you know from the structures we have talked about in the other presentations this is the location of the peptide bond.

    01:09 Now on the right side of this image, you can see the central part of a serine protease. Now the central part is the place here where the reaction is going to be catalyzed.

    01:22 Now it's a little hard to get our head around at some of these things.

    01:25 So you are gonna see in some cases, I am gonna stretch bonds and stretch molecules a little bit to actually make things fits so you can understand this.

    01:33 Please understand that in an enzyme itself, of course, they are already better positioned, but it's hard with figures to make things fit as we would like to. Serine proteases all have a common feature of their active site. And the common feature that they have of their active site is that they all contain these three amino acids side chains that you can see located in close proximity of each other.

    01:55 Now I always like to remind students that when we see something like this it reminds us that protein folding does occur.

    02:04 That is the serine and histidine and the aspartic acid which are the three side chains that we see here are not located close to each other in primary sequence.

    02:10 They are brought into close proximity of each other by the folding of the enzyme to make them physically close to each other, as we see here.

    02:22 And the closeness of these is important to start.

    02:25 But more importantly, the flexibility of the enzyme with these side chains is absolutely essential to the catalytic function that will happen.

    02:34 Okay. So we imagine now that we see this folded enzyme and then the rest of the enzyme is shown in yellow. We are looking right now specifically at the active site. Near the active site we have a place where the protein is going to bind, and the protein that's gonna be cut is going to be interacted with this catalytic triad of serine, histidine and aspartic acid.

    02:56 The binding of the substrate to the enzyme occurs in a specialized site on the enzyme call the S1 pocket.

    03:05 So we have shown here the S1 pocket that is a sort of a semi circle that's holding on to a part of that protein.

    03:12 We can see the protein that is going to be cut now is at the active site.

    03:17 Now in the binding of this protein to the active site you notice that the nitrogen on the histidine has an arrow pointing towards the hydroxide. We also note that the oxygen, that is on the side chain of aspartic acid has a little dot next to the hydrogen on the histidine.

    03:37 What's happened here? Well, then going from the previous slide to this slide, we can see that what's happened is the enzyme has changed shape very slightly.

    03:48 The binding of the substrate, and remember that binding of substrate changes enzymes, has changed the enzyme very slightly.

    03:56 So that the proximity of aspartic acid's side chain to histidines has changed.

    04:03 That's very important. Aspartic acid here, the oxygen has a negative charge, and the negative charge has moved a little bit closer to the ring of the histidine as shown here.

    04:15 By this small action, the electronic configuration of the ring of histidine is changed.

    04:23 And it's that change which is causing now the nitrogen to be reaching out and what it's going to do is it's going to grab that hydrogen that's on serine, okay? So this tiny change in shape that happen on the binding of the enzyme is starting the process by which the reaction is going to occur.

    04:41 So we can see here that the S1 pocket has facilitated all this happening.

    04:46 I should say in the S1 pocket, that the S1 pocket gives the specificity of the enzyme.

    04:54 The S1 pocket will not bind to everything.

    04:57 It will bind to specific proteins with specific sequences within them.

    05:03 Very very important concept. If it doesn't encounter those specific things, it won't bind them and if won't bind them, of course, there is nothing to react.

    05:10 At the end this process will not occur. Okay. So the slight chart structural changes have happened and we now see the result of this starting to come into play. The things, the entities have moved closer into each other. The electronic environment has definitely changed by this point.

    05:29 And what we see is that that proton that was on the OH of serine is now associated with the nitrogen of the histidine ring. Now this is the first step in this catalytic process. Actually the second step if we count the binding of the substrate.

    05:45 This making of the oxygen with a negative charge on the end of serine is fundamental to this reaction occurring.

    05:54 We call this negatively charged oxygen on serine, an alkoxide ion. Okay? That alkoxide ion that's on serine is extraordinary reactive.

    06:04 It's ready to go do business.

    06:07 Now we have stretched that S1 pocket little bit to remind us that again we are bringing things into closer proximity and that is important because the alkoxide ion is looking for something to bind to. It is looking for a nucleus. It's what we call a nucleophile.

    06:25 And the nucleus that it is looking for here is this carbon, which is the arrow that's being pointed from the oxygen minus down to the orange carbon.

    06:35 So there is actually what's called a chemical attack, a nucleophilic attack, that's occurring on that carbon.

    06:43 We can see that the electrons that are double bonded to the oxygen are rearranging, as we see, the arrow being pointed.

    06:51 And in the next step of the process what will happen is that we are going to see a rearrangement in the molecule.

    06:58 Okay? So we went from this position to this position. Notice that we had a carbon with a double bond to an oxygen that now is a carbon with a single bond to an oxygen.

    07:11 That molecule is chemically unstable.

    07:12 It's chemically unstable and a chemically unstable molecule has to be dealt with, because if it's not dealt with, it's going to cause problems.

    07:21 Well, the enzyme has another pocket in it to deal with that unstable molecules called the oxyanion hole.

    07:28 And the oxyanion hole helps that unstable molecule to fall apart without problem. That's pretty cool.

    07:36 Okay? It's going to fall apart without problem and what's gonna happen here as you can see is the nitrogen in blue is going to reach up and grab that hydrogen that was originally grabbed by the histidine side chain, okay? So this intermediate that's in the oxyanion hole is what we call a Tetrahedral, okay? And tetrahedrals we know from organic chemistry are what happens when carbon has those four bonds that you can see here, okay? The peptide bond which is between the carbon and the nitrogen, is going to be the broken as a result of nitrogen grabbing that hydrogen.

    08:13 Here, nitrogen has grabbed the hydrogen. The grabbing of the hydrogen from the histidine cause the bond between the carbon and the nitrogen to break.

    08:23 So we have broken the peptide bond .And so part of the protein, the part of the protein shown in blue, is now free to go and do it's business. It's released.

    08:32 There is nothing attaching it to the enzymes and it goes and it exits.

    08:35 What we have done here is we have actually gone through the first part of the reaction.

    08:42 And in this part of the reaction is what we call the rapid part of the reaction, okay? The other part of the protein is attached to serine.

    08:51 It's physically attached to serine. It's a covalent bond at this point.

    08:55 Now that covalent bond has to be broken in order for the other part of the original protein to be released.

    09:04 And that's what gonna happen in the slow step of catalysis.

    09:08 Now the slow step of catalysis actually has about the same number of steps as the fast step of catalysis.

    09:14 But other things have to happen including the movement of water into the active site, in order for this peptide to be released.

    09:23 Well, we see that happening here. Water now has physically moved into the active site. There is a molecule of water. And that process that we saw of the nitrogen on histidine taking a proton is going to repeat itself.

    09:36 We see it happening here. We see the arrow from the nitrogen on the histidine pointing to the hydrogen on water.

    09:43 So it's gonna take that hydrogen instead of taking the hydrogen that is originally took, which is no longer there, on serine.

    09:49 What's gonna happen in that process is now we are gonna have an activated oxygen like we had with the alkoxide ion except for here it's gonna be a hydroxide.

    10:00 We are gonna have an activated oxygen that is gonna make a nucleophilic attack on carbon just like we saw before.

    10:07 So there is a nucleophilic attack that's going to happen in the process of this moving forward.

    10:13 Here is the attack of the hydroxide and look what happens. We see that the electrons on oxygen are going to rearrange.

    10:22 We create a tetrahedral immediate as we created before. And now there is the oxyanion hole stabilize in that intermediate.

    10:33 We now see that what happens is that oxygen is going to attack the hydrogen on that group and it's gonna pour away just like the first peptide did.

    10:42 When it does that, what happens is the molecule released. So we see the second half of the polypeptide chain released and in addition the enzyme returned back to it's original state. Gone and as it were.

    11:03 The cycle is now complete. So it is about 10 steps going through what I described here and the important thing to understand about this is that the enzyme started at one state.

    11:13 It went through a transition and then went back to the original state it was in. Very much like the process I have already described but now you have seen it in mechanistic terms.

    11:22 When we saw the image of the reaction occurring, we saw these various states that you see on the screen.

    About the Lecture

    The lecture Serine Proteases – Enzyme Catalysis by Kevin Ahern, PhD is from the course Enzymes and Enzyme Kinetics.

    Included Quiz Questions

    1. It lowers the energy of activation of a reaction
    2. It changes the Gibbs free energy change of a reaction
    3. It changes the standard Gibbs free energy change of a reaction
    4. It only allows reactions to go forward
    1. They use serine for catalysis in their active sites
    2. They cut target proteins at serine residues
    3. They bind serine during catalysis
    4. They are misnamed because they do not involve serine at all
    1. Serine, histidine, and aspartic acid
    2. Serine, glutamic acid, and histidine
    3. Serine, glycine, and aspartic acid
    4. Serine, lysine/ and histidine
    5. Serine, alanine, and selenocysteine
    1. Oxyanion hole
    2. Active site
    3. Allosteric site
    4. S1 pocket
    5. Hydroxide/alkoxide pocket
    1. S1 pocket
    2. oxyanion hole
    3. active site
    4. allosteric site
    5. alkoxide/hydroxide pocket
    1. Alkoxide ion produced on serine
    2. H+ ion
    3. N+ of histidine ring
    4. H+ of serine
    5. O- of aspartic acid
    1. Aspartic acid and histidine play important roles.
    2. An electrophilic attack is at the core of the reaction mechanism.
    3. The slow step is the first one.
    4. The alkoxide ion forms on aspartic acid.
    1. It determines the binding/cutting specificity of the enzyme.
    2. It stabilizes an unstable tetrahedral intermediate.
    3. It is the location of the catalytic triad.
    4. It requires serine for binding.

    Author of lecture Serine Proteases – Enzyme Catalysis

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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    Well explained
    By Mary T. on 26. January 2019 for Serine Proteases – Enzyme Catalysis

    Attaractively and well explained video of serine protease catalytic mechanism that I have ever seen...Thank you..????

    Thanks a lot prof.
    By Ahmad Jawid A. on 30. April 2018 for Serine Proteases – Enzyme Catalysis

    It is really helpful. I appreciate your kind efforts. Thanks verh much.

    By Amna A. on 26. April 2018 for Serine Proteases – Enzyme Catalysis

    He simplified the concepts and illustrated by a very nice way