Understanding Proteases: Mechanisms of Serine, Cysteine, Aspartate, and Metalloproteases

so far in our discussion on protease is we focused on serine proteases and we discussed three examples of serum

protease so we discussed chymotrypsin trypsin and elastase now we said these three Seor proteases all use the same

catalytic triad so in their active site they have this collection of three amino acids that work together to basically

promote the hydrolysis of peptide bonds and out of these three amino acids it's the serine that acts as the

nucleophilic agent now what about the other categories of protease is so remember we not only have serine

proteases but we also have cysteine protease as a spritzel proteases metalloproteases and we have other

examples of proteases that we're not going to focus on in this lecture so the question is what exactly is the

mechanism of these other proteases and how does the mechanism differ or how does it compare to the mechanism inside

serine proteases so let's begin by briefly discussing cysteine proteases so in cysteine proteases we also have

amino acids in the active side that work together to basically catalyze the cleavage of peptide bonds but unlike

in-ceiling protease is insisting proteases it's the cysteine residue found in the

active side that acts as that nucleophilic agent that will cleave that peptide bond and to see how that

actually works let's take a look at the following diagram so we have a cysteine residue in the active site of some

particular cysteine protease now in the form that we have cysteine here cysteine is not good it is not a strong enough

nucleophile so the sulfur as shown here is not a strong enough nucleophile and it will not be able to attack the carbon

of that carbonyl group on that substrate molecule because this simply isn't a good enough nucleophile so what must

happen is another residue must work together with the same to basically transform it into a better

nucleophile so how does that actually take place well in a similar way to how takes place in serine protease is so we

have a nearby residue for example a histidine and on that histidine we have this nitrogen which contains a lone pair

of electrons on top of that the nitrogen can also have or also has a partial negative charge because it's more

electronegative than the nearby carbon atoms now if we examine this H on the sulfur we see that the H contains a

partial positive charge because the sulfur is more electronegative and the sulfur contains a partial negative

charge so what we see happening is before the substrate actually enters the active side what begins to happen is the

nitrogen because of its higher electron density begins to pull on this h ion and as the H ion is being pulled away on

center nitrogen as shown in this particular diagram these two electrons in the Sigma bond move closer to the

sulphur atom and that increases the electron density the electron cloud around the sulphur and that increases

its ability to act as a nucleophile so as the H atom is being pulled away onto the nitrogen of the nearby histidine

residue we see that these two electrons move closer to the sulfur and the electron density on the sulfur increases

and so as we have this incoming substrate molecule these two electrons attack nucleophilically the carbon on

the carbonyl and that displaces the PI bond and so once the step takes place we form the same type of tetrahedral

intermediate that we formed in the serine protease reaction mechanism and just like in serum protease as we can

have an oxyanion hole that stabilizes the negative charge on that oxygen once we formed a tetrahedral intermediate in

Syst protease is we can also have a similar type of oxyanion hole that stabilizes

that relatively unstable and high-energy tetrahedral intermediate now of course eventually the tetrahedral intermediate

will collapse and after a few processes were going to basically break that peptide bond in an in a similar way to

how we broke the peptide bond in serine proteases now two examples of cysteine proteases are caspases these are those

enzymes involved in the process of programmed cell death so abbott osis as well as cathepsins which are involved in

immunity now let's move on to aspartate protease is also known as a spritzel proteases now the major difference

between cysteine proteases and aspinall proteases is the following insisting proteases as well as the serine

proteases we see that one of the residues inside the active side of the enzyme acts as a nucleophile but in a

special protease the residues don't actually act as nucleophiles instead it's the water that will ultimately act

as the nucleophile now the similarity between let's say cysteine protease and a special protease

is we still have to transform the water into a good nucleophile in the same way that we have to transform the cysteine

into a strong nucleophile before the nucleophilic attack took place in this particular case we see that we're going

to have residues that will transform the water into a better nucleophile now if we examine the active site of a special

proteases we're going to find two residues one of these residues will be aspartic acid and the other one will be

aspartate so this is basically the same thing as this but this is in the deprotonated state and this is in the

protonated state so we have aspartate we have aspartic acid now when water moves into the active side the

negative charge on the aspartate will interact with the positive charge on one of the H atoms so we have a partial

positive charge here because the oxygen contains a partial negative charge and so by the same by the same analogy here

when the nitrogen pulls away the h it gives the electrons in the sulphur and so the electron density on the sulphur

increases and it becomes a better nucleophile here when the negative charge the oxygen pulls away the Aged

it makes the oxygen a better nucleophile because we essentially transform water into a hydroxide and the hydroxide is a

much stronger nucleophile at the same time what happens is the other residue the sponeck acid uses its H to basically

interact with the oxygen of the carbonyl so the oxygen here contains a partial negative charge and the H here contains

a partial positive charge and so as they interact here what begins to take place is we basically make the carbon of the

carbonyl a better electrophile so the ultimate reason why we have these two different we have the aspartate and the

spartax acid is because one transforms the water into a better nucleophile and the other one transforms the incoming

substrate into a better electrophile and now we can have our reaction take place the similar reaction the same type of

reaction that took place here we have this bond breaking forming a bond with carbon attacking the carbon

nucleophilically displacing the PI bond forming that same type of tetrahedral intermediate except now instead of

having a bond between this residue and the carbon we have a bond between the oxygen of the water and this carbon so

inside the active side of these proteases is a pair of aspartic acid residues they work together to transform

water into a good nucleophile so that it can hydrolyze that peptide bond so what happens is

deprotonated aspartic acid that aspartate shown here uses its negative charge to transform the water into a

better nucleophile so we essentially create a hydroxide and then the protonated version of the residue the

sparta castle uses the partially positive hydrogen to basically interact with that partially negative oxygen of

the carbonyl and that creates a good electrophile and now the reaction can take place at a relatively high rate now

two examples of aspartate proteases that we're going to focus on in future lectures is renin and this is basically

the enzyme that is used to control blood pressure as well as pepsin and this is one of the digestive enzymes that is

found in our digestive system and finally let's take a look at metalloproteases so just like the name

implies this has to do with the fact that inside the active site of metalloproteases we actually have unmet

'el atoms and usually the metal atom is zinc so what's the point of the metal atom well for the same exact reason that

we have this residue to basically transform the water into a better nucleophile here we also have a metal

atom to actually bind the water and transform it into a better nucleophile so that the water can basically

hydrolyze that peptide bond so if we examine the active site as shown here let's say we have the zinc metal atom

that is actually down to the active side of that enzyme and so what it does is it interacts with the oxygen as a result of

this being partially negative and this having a positive charge so they form this bond at the same time some type of

nearby residue acts as a base and usually the residue is glutamate so this acts as a base and basically grabs off

the Aged it pulls the H away from this oxygen and that transforms the oxygen into a much better nucleophile so it

creates that hydroxide and this can act as a nucleophile attacking the carbon of the carbonyl and once

again forming a tetrahedral intermediate and then following some several steps we basically collapse and break apart

that tetrahedral intermediate and eventually we hydrolyze that peptide bond now one example of a

metalloproteins that is found inside our body is carboxy peptidase a and this is once again another example of a

digestive enzyme so we see that even though we have many different groups many different types of protease is we

have serine proteases cysteine protease --is a special protease --is metalloproteases and so forth all these

different types of proteases basically function in a very similar way what they ultimately do is they transform a bad

nucleophile into a good nucleophile and they transform a bad electrophile into a better electrophile and so that

ultimately we have a strong nucleophile attacking a very good electrophile and that's exactly what increases the rate

of that the rate of that reaction the rate of the hydrolysis of that peptide bond