Understanding Kimotripsin: The Sering Protease's Catalytic Mechanism and Specificity

kimot Tron is a sering protease and it has the ability to catalyze the cleavage of peptide bonds on the carboxy end of

large hydrophobic non-polar amino acids for instance kimot Trion Cleaves at the carboxy end of methionine phenol alanine

tyrosine as well as tryptophan now as we discussed previously it's the presence of the catalytic Triad inside the active

side of kyrion that actually gives it the power of catalysis gives it the ability to actually cleave those peptide

bonds so remember the catalytic Triad is basically this collection of three individual residues aspartate histadine

and cine which work together to basically promote the cleavage of those peptide bonds now the question Still

Remains what exactly gives kyrion its specificity what gives Kim trips in the ability to only cleave on the carboxy

end of specific amino acids those amino acids that contain bulky hydrophobic side chain groups now to answer this

question we actually have to study the shape of the active side the structure of that enzyme if we examine the active

side of that enzyme we're going to find something called the S1 pocket and the S1 pocket in kimat tripid is basically

that real region to which that side chain group will actually move into and if we examine the shape and structure of

the S1 pocket we're going to find that it's relatively long so relatively deep and mostly hydrophobic so non-polar and

because of that structure of the S1 pocket only those amino acids that contain side chain groups that are long

nonpolar do not have any charges will actually be able to fit into that pocket into the active side without creating

too much electric repulsion so amino acids such as methine phenol alanine tyrosine and tryptophan so once again

it's the catalytic Triad in the active side it's the presence of these three individual amino acids that give kimot

Tron its catalytic power the ability to actually catalyze the cleavage of those peptide bonds but it's this long and

narrow shape and the fact that it's mostly hydrophobic it's the shape of the S1 pocket that actually gives katriin

its specific nature its specificity to cleave only on the carboxy and of specific side chain

groups now as we discussed in our uh study of proteases we basically said there are many different types of

proteases that exist in inside our body and inside nature now so far we focused on seing proteases and we use kimot

tripson as the prototypical sering proteas but of course in our body in our digestive system for example we have

many other examples of searing proteases the question is what is the mechanism that these other seing proteases

actually Ed to cleave peptide bonds well it turns out other serum proteases also use this same catalytic Triad and what

that means is they also carry out the catalysis process by using the same exact mechanism namely Cove valent

catalysis and acidbase catalysis and two examples of other serum proteases inside our digestive system that also contain

the same catalytic Triad is Tron as well as elastase so kimat Trion is is not the

only sering protease that utilizes this catalytic Triad in fact Tron and elastase are two other sering proteases

found in our digestive system that use this same exact catalytic Triad and therefore the same exact mechanism of

catalysis that we spoke about in the previous lecture now the question is why do we have different types of CM

proteases inside our body body and the question is what exactly differentiates tripson elastase and kimat Trion if they

have the same exact catalytic Triad well remember it's the catalytic Triad that gives the enzyme its catalytic power

that gives the protease the ability to cleave those peptide bonds but it's the shape of that active side the S1 pocket

that determines the specificity of that protease the type of eptide Bond it actually breaks and so the difference

between Kima trips and tripson and El lastas is not in the type of catalytic Triad used but it's in the shape of that

particular S1 pocket the structure of that S1 pocket as we'll see in just a moment as a result of a slight variation

in the S1 pocket of tripson and Al last days we see that these other serm proteases cleave other amino acid so all

the trips in El last days use the same mechanism so coent catalysis and acid-based catalysis which we spoke

about in the previous lecture they differ in their specificity and that has to do uh and that has to do to the fact

that there is a slight structural difference in the S1 pocket in tripson as well as alasas and let's see what

these differences are and what they result so let's begin with Trion so Tron catalyzes the cleavage of peptide bonds

on the carboxy end of Lysine and Arginine and if you recall lysine and Arginine both contain positive charges

on their side chain groups now the question is why is this true so tripson uses the same exact catalytic Triad that

kimat Trion uses but what gives this Tron the difference in specificity well if we examine the S1

pocket of tripson at the bottom of that S1 pocket we're going to see we're going to see a residue that we don't see in

the S1 pocket of Kima tripson at the bottom of the tripson S1 pocket we have a negatively chared side chain that came

from the aspartate that we see in tripson and we don't see in Kima tripson so as a result of the negatively chared

side chain of aspartate 189 that is found at the bottom of the S1 tripson pocket we see that this tripon only

Cleaves at the carboxy end of those amino acids which are long and contain a positive charge at the end and these

happen to be lysine and Arginine so if we examine the following hypothetical polypeptide we have glycine this one

here we have lysine this one here we have glycine this one we have Arginine this one and we have glycine this one

now the only ones that contain positive charges are these two amino acids and only these amino acids will be able to

actually fit into the pocket of Tron and at the end will be able to actually interact in a stabilizing manner so the

positive charges of these two side chain groups will interact with the negative charges of of the of this aspartate 189

and that and that will create a stabilizing effect it will neutralize the net charge in that space and that

will create a very stable effect so at the bottom of the active side of tripson is an aspartate residue and the negative

charge of the aspartate side chain group will stabilize those amino acids that contain side chain groups with positive

charges namely the lysine and the Arginine and so the only bonds that tripson will be able to cleave are this

Bond and this Bond here so at the carboxy end of Lysine and Arginine so we see that a very tiny

variation in the structure of the S1 pocket in tripson gives tripson uh a different specificity than that of kimat

Trion and finally let's move on to AAS days so if we examine the S1 pocket of elast days we'll see the presence of two

additional valine and valine if you recall are these small or valine molecules have these small relatively

small hydrophobic chains and notice where these two valins are positioned they're positioned opposite of each

other and they essentially play the role of blocking the majority of the bottom portion of that S1 pocket and so what

that means is when the side chain group of the amino acid moves into the S1 pocket it cannot actually occupy this

space here because this blocks as a result of the stair Kinder of the hydrophobic properties of these side

chain groups and so only those amino acids that have relatively small side chain groups and which are non-polar so

uncharged will be able to fit into this pocket and so we see that elastase Cleaves peptide bonds on the carboxy end

of small hydrophobic amino acids such as glycine valine alanine valine alanine Lucine and isoline as well as Seine so

if we have this hypothetical polypeptide chain we have glycine lysine alanine phenol alanine glycine so the only ones

which have a small hydrophobic side chain are glycine alanine as well as glycine at the end so

the only two peptide bonds that are going to be cleaved are this peptide bond so on the carboxy end of glycine as

well as this one on the carboxy end of alanine so lysine and phenol alanine are not small ones this one has a large long

one and it's positively charged although this one is is uh non-polar hydrophobic it's too large to actually fit into this

pocket because these two valins block the majority of that pocket and even though this is a glycine there's no bond

on the car boxal side and so nothing will be cleaved on this side by last days and so we see that two Valiant

residues found in the S1 pocket of el lastes block off the majority of the pocket in El lastes and this allows the

last days to only cleave small hydrophobic residues and so we see that the majority of the serum proteases

found inside our body for instance kimat Trion Tron as well as elastase although they use the same catalytic Triad to

basically catalyze the cleavage of the peptide bonds they actually differ in the types of amino acids that they

cleave types of peptide bonds that they cleave as a result of these structural differences and shapes in the S1 pockets

of their active sides