Understanding the Catalytic Triad in Chymotrypsin: Mechanism of Peptide Bond Cleavage

if we examine the active side of kimot Tron we're going to discover a collection of three different residues

three different amino acids that act together that work together to promote and catalyze the cleavage of peptide

bonds and this collection of three amino acids is known as the catalytic Triad so it's the catalytic Triad inside the

active site of kimat tripson that essentially catalyzes the cleavage of peptide bonds now this catalytic Triad

consists of three different amino acids one of these amino acids is cerine so cine 195 the second amino acid is

histadine 57 and the third amino acid is aspartate 102 so let's begin by discussing the role that each one of

these amino acids actually plays in promoting the hydrolysis of peptide bonds well let's begin with Serene 195

we know that kyrion is an example of a searing protease and so what that means is it's ultimately the Searing residue

inside the active side it's this residue here that acts as a nucleophile and will attack the carbon of the carbonal that

peptide bond nucleophilically and we'll see exactly how that works out in just a moment now the problem with cine in this

form shown here is the side chain of Seine is in its alcohol form and we know from organic chemistry that alcohols

aren't very good nucleophile so the problem here is this alcohol is not a strong nucleophile and in the form we

have it now it will not be good enough nucleophile to attack that peptide bond and so what must happen is the histadine

and the aspartate must work together to transform this searing into a strong nucleophile so what we essentially want

to do is transform alcohol into its conjugate base the AL oxide because remember Al oxide molecules Al oxide

ions have a much better ability to actually act as nucleophiles because they have a better electron density

around that oxygen atom so what happens is the negatively charged side chain of aspartate basically interacts with this

partially positive hydrogen atom so if we examine so where is the color red so let's take

red and blue so if we examine the charge value on this hydrogen because the nitrogen is more electron negative than

the hydrogen what that means is this H atom will bear a partial positive charge and so these two side chains will

basically interact as shown in this diagram we have Electro electromagnetic interaction between the oxygen and the H

and what this does is it basically positions this entire side chain of histadine 157 in the correct orientation

so that the next interaction can take place now what exactly is the next interaction well this nitrogen contains

two electrons a lone pair of electrons on top of that we can also say that the nitrogen has a partial positive charge a

partial negative charge because nitrogen is more Electro negative than the near by carbons and so we can say there is

this partial negative charge that exists on this nitrogen and so it will interact with the H atom because this H atom of

this alcohol of the cine contains a partial positive charge because this oxygen contains a partial negative

charge because of its high electro negativity value and so aspartate uh aspartate 102 basically interacts with

histadine 57 to move it and position it into the correct orientation so that the lone pair of electrons on the nitrogen

can now interact and pull away this H atom now once the H atom is pulled away that transforms that alcohol group into

an into an Al oxide group and because the AL oxide is a much stronger nucleophile this will now interact with

the carbon of the carbon and essentially break that peptide bond as we'll see in just a moment so we see that the cine

195 in its alcohol form is simply not a strong enough nucleophile and to transform it into a better nuclear file

a nearby histadine 57 pulls away the hydrogen ion to form an Al oxide and to actually position the histadine so that

these two residues can interact very well this asate uses its full positive charge to basically position and move

this histadine side chain in the correct orientation and together this catalytic Triad as we'll see in just a moment

actually promotes the cleavage of the peptide bond so let's actually discuss what the

reaction mechanism is so what are the details of the reaction mechanism that takes place inside the active side of

kimat triin so let's begin in the following stage so we have the aspartate 102 that positions this histadine 57 so

that these electrons can interact with the H atom and so they begin to pull away the H atom and as the H atom is

being pulled away this is being transformed into an Al coxide and the AL coxide is a strong enough nucleophile it

contains a high enough electron density around the oxygen as to actually attack nucleophilically this carbon of this

peptide bond and so once the carbon is attacked that displaces the pi Bond and places those two electrons that were in

a that were initially in the pi Bond onto this oxygen so the seran AL oxide acts as a nucleophile and attacks the

carbon of the carbonal and what we ultimately form after Step One is we form a tetrahedral in intermediate so in

this particular case if we examine this Bond here we see that we have SP2 hybridization and what that means is

this is going to be a planer molecule and that gives this molecule stability but as soon as this attack takes place

we form a tetrahedral intermediate and on on top of that we're going to have a negative charge on this oxygen and this

tetrahedral intermediate because of that negative charge and because this molecule is no longer planer it's not

going to be a stable so in step one we formed the relatively unstable tetrahedral intermediate so we call it a

tetrahedral because here we have one two three sigma bonds and here the carbon has 1 2 3 4 Sigma bonds now because of

the instability of this intermediate a special pocket a special region on the kyrion enzyme known as the oxyanion hole

or oxy anine pocket basically interacts with the negative charge on this oxygen so inside the pocket we have these

nitrogen atoms that contain H atoms and these partially positive H atoms can interact with the fully negative oxygen

atom and so what the oxy anine hole does is it stabilizes this tetrahedral intermediate now because of the

instability of the tetrahedral intermediate it's not going to exist for a very long time and what that means is

it's going to very quickly collapse and when a collapses what happens is the lone pair of electrons on the oxygen

forms a pi bond with this carbon and that breaks off this relatively weak nitrogen Bond and so when this Bond

breaks off the electrons on those Bond on that Bond basically move on and grab this H atom because by grabbing the H

atom away from this histadine this loses that positive charge and becomes more stable and that can be seen in this

diagram here so after step two after this tetrahedral intermediate collapses that essentially asalat this Seine

residue so this AEL group is now attached onto this oxygen and this amide has been formed and the amide takes away

this H Atom from the nitrogen found on this side chain of histadine 57 and so now we have this slide interaction

between the nitrogen and the hydrogen and we still have the interaction between the oxygen and this hydrogen so

this ulates the serine and forms an amine molecule that deprotonates the his nitrogen shown here and once we form

this amide product in The Next Step the amide basically moves away and when the amide moves away we basically make room

in the active side for a water molecule to actually enter because remember it's the water molecule that will ultimately

also act as a nucleophile to basically help hydrolize that peptide bond so in the next step once the amide product

departs we have the water molecule that comes into place and so this water molecule it basically positions itself

into the same position that we had this amide in this step and once it positions into this location the lone pair of

electrons on the nitrogen of this side chain of the histadine basically interact with this H and they

deprotonate that uh that water molecule now this is a very important step because just like Seine contains the

alcohol and the alcohol is not a strong enough nucleophile so what happens is the H atom is removed to create the AL

oxide and transform into good nucleophile in this case water is also not a strong enough nucleophile to

actually attack the carbon of this double bond and so what must take place is again we see that the histadine

actually takes away the H from this oxygen and that transforms the water into a hydroxy and the hydroxide is a

good enough nucleophile so remember from organic chemistry that hydroxide molecules contain a full negative charge

on the oxygen and so that makes it a strong nucleophile and so this nitrogen takes

away the H atom and these two electrons that were in the sigma Bond now nucleophilically attack the carbon and

this displaces the pi Bond in the same way that we displac the pi Bond here and in the same way that we

form this tetrahedral intermediate we also form a tetrahedral intermediate in step five and once again to stabilize

that relatively unstable and negatively charged tetrahedral intermediate we have this oxy iion uh oxyanion hole that

contains the partially positive charge H atoms that can stabilize this full negative charge and so because it's so

unstable it doesn't exist for a very long time what and what happens is again the two electrons on the oxygen

basically form a pi bond between the carbon and this oxygen and now what happens is this Bond here shown in green

that we basically formed in this step is now broken and when this Bond breaks the two electrons that existed in that Sigma

Bond now basically move on to this H atom and take away that H atom and again the reason we want to take away that H

atom is because we want to remove this positive charge that exists on the Ring of this histadine 57 side chain and so

in the next step we basically reform that alcohol group found on the cine we remove that H atom that was on the

nitrogen so we reform form the histadine 57 side chain and we also form this final product the carboxylic acid and so

now this is one of the products this is the other product and together we see that the end result is the cleavage of

that peptide bond so this bond between the nitrogen and the carbon was essentially cleaved in this step as

described here and so in the final step this carboxilic acid product basically departs it leaves the active side and

once it leaves the active side it basically prepares the active side for another cycle of hydrolysis so this is

the reaction mechanism that actually takes place inside the active side of kimot Trin and so the important point

about this mechanism is inside the active side we have this catalytic Triad this collection of amino acids

which basically work together to create a strong nucleophile and by creating a strong a a strong nucleophile they

essentially allow the hydrolysis the catalysis of the hydrolysis of peptide bonds and notice that we create a strong

nucleophile not only in this case where we transform the alcohol into an Al oxide but we also transform the water

molecule a poor nucleophile into hydroxide into a much better nucleophile in step four so we see that in the

reaction mechanism this catalytic Triad basically acts twice to transform a poor nucleophile into a strong enough

nucleophile to actually nucleophilically attack that peptide bond and ultimately break that peptide bond