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chymotrypsin is an enzyme that cleaves
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after aromatic residues such as
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phenylalanine tyrosine or tryptophan
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within the active site of chymotrypsin
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we see a hydrophobic pocket
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an oxyanion whole
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a glycine residue
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and three different amino acids over
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here
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now these three amino acids aspartate
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102 histidine 57 serine 195
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form a catalytic triad
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meaning that these three amino acid
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residues work together in the active
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site in order to cleave our peptide bond
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after the aromatic residue
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now what do these numbers mean at the
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top of each of these amino acids
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this number represents the location at
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which this amino acid occurs
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so we know that
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when we have amino acid chains we have
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lots of amino acids so aspartate occurs
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at position number 102 whereas histidine
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occurs at position number 57 and serine
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at 195.
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now we also have this glycine residue
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which is actually going to be
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stabilizing our oxyanion
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which we'll see later in the steps
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now the hydrophobic pocket is where our
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hydrophobic group is going to sit
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so when we take a look at phenylalanine
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tyrosine and tryptophan they all have
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very bulky aromatics that are
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hydrophobic
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and these hydrophobic
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parts of these amino acids will reside
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over here and this in a way keeps the
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keeps the polypeptide in place while it
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is being cleaved
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now in the first step we add in our
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substrate which is our polypeptide
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and we add it into the active site
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when it enters its active site remember
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we are reading the polypeptide from left
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to right so we'll cleave it after the
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aromatic so we will cleave it at this
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peptide bond between the carbon and the
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nitrogen
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this is where we are going to cleave
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now if we take a look over here we can
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see that
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uh this this
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histidine is attacking the hydrogen on
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the serine
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now we have to think why is this
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happening this is happening because when
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the substrate enters the active site of
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chymotrypsin
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the enzyme chymotrypsin
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um goes through a little bit of a
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conformational change in order to fit
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the substrate and this is referred to as
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induced fit
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so when induced fit occurs and the
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substrate binds this compresses the bond
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between aspartate and histidine remember
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we have a bond right over here between
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aspartate and histidine
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the negatively charged oxygen is
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interacting with that hydrogen so when
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our substrate is added in this bond is
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actually compressed
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so normally histidine has a pka value of
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six
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okay that's our normal pka value but
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after this uh bond compresses
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the pka of histidine changes to over 12.
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and we know we know that pka it's
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the potential of
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dissociating
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donating our hydrogens
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but we know also know that our
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environment so the molecules or
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substituents that are surrounding uh
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surrounding our molecule are going to
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affect the
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the pka value so since this bond
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compresses it affects the histidine and
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now the pka has increased to 12. so
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since the pka has increased to past 12
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this makes histidine a very good base
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now and we know that bases the
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definition of a base is that
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it will
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be a very good uh proton acceptor
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so it will want a proton so that is why
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we see histidine over here abstracting
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that proton from serine because it's a
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very good base and it has that nitrogen
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has that lone pair so it's going to
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abstract that hydrogen
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but now this oxygen on the serine
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is going to gain a lone pair
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now this lone pair is now
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going to create a negatively charged
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oxygen which is going to
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nucleophilically attack this carbon over
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here now this carbon is part of our
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polypeptide which has entered the
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substrate
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so when this oxygen nucleophilically
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attacks this carbon we know that carbon
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can only have four bonds
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so what's going to happen is that carbon
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is going to break one of its bonds and
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it's going to be this
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one over here so oxygen is going to gain
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a lone pair okay and we'll see that on
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the next slide
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so here we can see that oxygen has
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gained a lone pair
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and it's gained a negative charge
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we can
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also see that the serine
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this oxygen remember that this oxygen
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just nucleophilically attacked this
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carbon in the previous step
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we can also see that histidine now has
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formed a covalent bond with hydrogen
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now what's going to happen is that
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this is a very short-lived intermediate
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okay
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this is the tetrahedral intermediate and
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this is very very interesting this is
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called the tetrahedral intermediate
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because now carbon has four bonds four
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different bonds before it only had three
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now it has four so this is our first
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tetrahedral intermediate
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and this is a very short-lived unstable
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intermediate because of this oxyanion
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now what's important to note over here
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is that this oxygen is actually inside
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that oxyanion hole we were talking about
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it's positioned in this oxyanion whole
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so it can interact with the hydrogen of
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glycine and the hydrogen of serine and
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they're interacting via hydrogen bonding
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which is stabilizing this oxyanion but
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it's still a relatively uh unstable so
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oxygen is going to regain that double
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bond it had with carbon and as it does
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so
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uh carbon can only have four bonds so it
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must lose another bond
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and it's going to be this one over here
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this uh this bond over here this
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structure over here is a very good
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leaving group
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so
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it's going to break off now as this bond
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breaks off this nitrogen over here is
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going to gain a lone pair
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and it's it's going to have that extra
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lone pair and nitrogen normally wants to
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have three bonds so as it's leaving
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it's going to abstract the hydrogen on
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that histidine
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so as it leaves we can see over here as
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it left it abstracted that hydrogen on
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the histidine
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and this is our first product now
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remember this was originally part of our
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polypeptide
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but now it has become our first product
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and this is what we are left with we are
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left with our acyl enzyme intermediate
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so
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once again we can note over here is that
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this oxygen has regained its double bond
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and we are still bound to serine
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now we have to break this part of our of
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our fragment that we are left with
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now in this step we can see that we have
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our acyl enzyme intermediate and what's
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gonna happen is that we're gonna add
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water now water is a great molecule to
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add in mechanisms because
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oxygen um has two hydrogens which can be
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abstracted now remember this histidine
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it still has that pka
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value greater than 12 so it's still a
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really good base
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because the bond between the aspartate
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and the histidine has been compressed
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and it's increasing that pka of
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histidine
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so the nitrogen on histidine is going to
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abstract that hydrogen and oxygen over
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here on the water is going to gain a
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lone pair and it's going to
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nucleophilically attack that same carbon
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again now once it does so we see the
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same thing happen this oxygen gains a
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lone pair gains a negative charge it's
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going to become an oxyanion within this
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oxyanion whole and we'll see in the next
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step that this charge is going to be
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stabilized with the serine and glycine
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once again
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so over here we see that
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the glycine and serine are stabilizing
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that negative charge we can see that
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histidine has abstracted that hydrogen
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from the water and now it has a covalent
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bond with hydrogen but now what's going
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to happen is that once again this
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oxyanion this is a this is our second
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tetrahedral intermediate because carbon
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has four bonds
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and once again the
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unstable oxyanion it's going to reform
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that bond that it had with carbon and
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remember that carbon can only have four
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bonds so it's going to lose a bond so
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now this is going to be our good leaving
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group and this bond is going to break
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so when this bond breaks this oxygen
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gains a lone pair and once it gains a
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lone pair it's more it wants to abstract
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its hydrogen remember that in our first
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step serine lost its hydrogen to
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histidine now it's coming back and
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saying hey i want my hydrogen back
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so it's going to abstract its hydrogen
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back
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now over here we can see that serine
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has abstracted its hydrogen
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and now we are left with this this part
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of our
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this part of
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our original polypeptide which is now
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going to break off and become
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our second product
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now
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um this oxygen is still in its oxyanion
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whole and we still see the
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phenylalanine inside that hydrophobic
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pocket which is keeping the entire
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molecule of the overall
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fragment in place
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now in the next step we can see over
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here this is the same depiction as the
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previous slide where serine has
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abstracted its hydrogen
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we're left with this part of the
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fragment and now this part of the
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fragment is going to be released from
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the active site and this is our second
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product and then we are back to our
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original state where we have our
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catalytic triad where they're
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interacting with one another oxy anion
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whole is empty again hydrophobic pocket
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is empty again and we are ready to go
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for another round of cleavage
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and that is essentially the chymotrypsin
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mechanism if we understand the first
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half the second half is exactly the same
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we end up with two products
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um we end up with two products and
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remember that enzymes do not get used up
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or changed in a reaction
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so whatever we start with that's what we
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must end with
