Understanding the Catalytic Properties of Enzymes: A Deep Dive into Kymotripsin
Heads up!
This summary and transcript were automatically generated using AI with the Free YouTube Transcript Summary Tool by LunaNotes.
Generate a summary for freeIf you found this summary useful, consider buying us a coffee. It would help us a lot!
Introduction
Understanding kymotripsin is critical for grasping the catalytic properties of enzymes. Kymotripsin is a serine protease found in the small intestine responsible for hydrolyzing peptide bonds, particularly those involving bulky hydrophobic amino acids such as methionine, phenylalanine, tyrosine, and tryptophan. This article will explore how kymotripsin functions within its active site, the significance of its structure, and the mechanisms that facilitate its enzymatic activity.
The Active Site of Kymotripsin
What is a Protease?
Proteases are enzymes that catalyze the breakdown of proteins into smaller peptides or amino acids by hydrolyzing peptide bonds. Kymotripsin, specifically, executes this function on the carboxy side of large, bulky hydrophobic side chains.
Structure of Kymotripsin
Kymotripsin exhibits a three-dimensional structure that facilitates its function. For example, within a hypothetical polypeptide chain of seven amino acids, including glycine, methionine, phenylalanine, tyrosine, alanine, tryptophan, and glycine, we can observe the interactions taking place among the side chains. Key residues within the active site such as the serine 195 act as pivotal catalytic components.
Key Amino Acids Targeted by Kymotripsin
- Methionine
- Phenylalanine
- Tyrosine
- Tryptophan
Those amino acids have bulky and hydrophobic properties, making them favorable substrates for kymotripsin's catalytic action. In contrast, smaller amino acids such as alanine or glycine are not cleaved as they do not meet the criteria of a suitable substrate.
Mechanism of Action
Covalent Catalysis
Kymotripsin employs a covalent catalysis mechanism, where a nucleophilic residue within the active site forms a temporary covalent bond with the substrate during the reaction, aiding in the cleavage of peptide bonds. The serine residue, specifically serine 195, plays a central role here. Its high reactivity allows it to interact with substrates effectively.
Inhibition Studies
To illustrate the importance of serine within the active site, scientists have utilized irreversible inhibitors such as diisopropyl phosphofluoridate (DFP). This inhibitor reacts with serine 195, thereby blocking the activity of kymotripsin. Such studies highlight the crucial role of serine within the enzyme’s active site, particularly since DFP only reacted with this specific serine residue among the 28 available in kymotripsin.
Two-Step Catalytic Mechanism
The reaction catalyzed by kymotripsin occurs in two main steps:
- Acylation - In the first step, the substrate binds to the serine 195, forming a temporary acyl-enzyme intermediate, which results in the release of an amide product and causes a rapid change in color measurable in spectrophotometry.
- Deacylation - This second, slower step involves the addition of a water molecule that cleaves the acyl-enzyme intermediate, regenerating the enzyme and yielding the final hydrolyzed product.
Observing the Reaction Progress
In experiments, changes in color and light absorbance are carefully monitored during the reaction. Initially, a steep increase in absorbance indicates a rapid substrate reaction due to the acylation step. As steady-state conditions are reached, the reaction rate slows, reflecting the slower deacylation step.
Graphical Representation of Reaction Progress
When plotting absorbance against time, scientists observe distinct phases of activity:
- Fast initial burst described by steep slope (rapid acylation)
- Slow steady-state described by leveling off slope (slow deacylation)
Conclusion
Kymotripsin exemplifies the intricate nature of enzyme catalysis, demonstrating how specific amino acids within active sites play crucial roles in enzymatic function. Understanding the mechanics behind kymotripsin's hydrolysis of peptide bonds is essential in biochemistry, highlighting how subtle molecular interactions lead to significant biological processes.
Future lectures will delve deeper into the specifics of the reaction mechanisms and catalytic strategies employed by enzymes like kymotripsin, offering a more granular view of how these molecular machines operate at the atomic level.
in order to understand the catalytic properties of enzymes we have to actually understand what takes place
inside the active sides of enzymes and to gain a better understanding as to what takes place inside the active sites
we're going to study a specific example of a protease that is found inside our body namely kyrion so kyrion is actually
a searing protease that is found inside the small intestine which basically hydrolyzes or catalyzes the hydrolysis
the cleavage of specific peptide bonds it Cleaves on the carboxy side of relatively large and bulky hydrophobic
side chain groups and this includes methionine pheny alanine it also includes tryptophan and tyrosine so if
we take a look at the following hypothetical polypeptide chain that consists of seven amino acids we have
glycine methine phenol alanine tyrosine alanine tryptophan and Glycine and this is the corresponding three-dimensional
structure now let's take any one of these amino acids so let's suppose this amino acid here and this happens to be
the tyrosine because this is the side chain group of tyrosine now this CC carbon here is the central carbon atom
this side is the carboxy side so the right side is the carboxy side and this is that carboxy peptide bond while the
other side is the amino side and this is that Amino peptide bond and what kimat Trion does is it only Cleaves methine
phenyalanine tyrosine and tryptophan on the carboxy side on the right side of that amino acid so if we examine let's
say methine so this is our side chain group of methine this is the green bond that will be cleaved by the kimat Trion
likewise if we move on to pheny alanine this is the pheny alanine so on the right side the carboxy side is where
kimot Trin will cleave that Bond again it's shown with green let's move on to tyrine this is tyrine this is the bond
that will be cleaved here and finally trip thean so Kima trips and Cleaves on this side of that Bond now let's take a
look at alanine well because alanine is not a bulky hydrophobic or aromatic amino acid what that basically means is
this Bond will not be cleaved by kimat tripson likewise this Bond will also not be cleaved because glycine is not a
bulky hydrophobic or aromatic amino acid so katriin is a Seine proteas that catalyzes the breaking the cleavage of
acids now katriin is a sering protease and katriin is an example of an enzyme that utilizes
the coent catalysis mechanism and what that means is inside the active side of katriin we have some type of
nucleophilic residue that acts as the Catalyst in forming a Cove valent Bond a temporary calent bond between the active
side of the enzyme and that peptide subst molecule now we say that the Cove valent bond is temporary because at the
end of the reaction that bond is broken and that's important because we have to reform that unmodified enzyme we have to
regenerate that enzyme at the end of any enzyme catalyze react remember enzymes are never changed or depleted at the end
of the reaction they always have to be regenerated and that's why this bond is only a temporary calent Bond
now we said earlier that kyrion is an example of a searing protease and what that means is inside the active side of
that kimot trsin it's the Seine molecule the Seine amino acid that plays the role of the powerful nucleophile that
catalyzes the cleavage of that peptide bond but how do we know that the active side contains this Seine atom a searing
molecule that plays the the central uh Central role in catalysis well basically the way that science discovered this is
they probed the active side with a specific type of irreversible inhibitor so they used the specific group
inhibitor known as diisopropyl phosphofluoridate or dif and this is the same irreversible inhibitor
that we spoke about in our lecture on irreversible Inhibitors so if we take dif and we mix it with an enzyme and
inside the active site we have a sering molecule and if the reactivity of that serin molecule is quite High then that
dipf will basically form a Cove valent bond with that oxygen of the serine displacing the H and that will kick off
this fluoride atom and we form a calent bond between the oxygen and the phosphorus atom and once we create this
coent modification the irreversible inhibitor basically blocks the activity of that enzyme and so we know that
because the activity of the enzyme is inhibited that means that the dipf must be bound to a Serene found inside the
active site now out of all the 28 Serene amino acids that we find in Kim tripson this is the only cine that reacts with
dif and this is found in inside the active side and so that implies that it's this searing molecule and the
Searing molecule has a number of 195 so if we count all the amino acids beginning with the first one along the
entire polypeptide chain this sering will be labeled as 195 so among the 28 Ser residues found
on kimat trips and only Serene 195 actually reacted with this irreversible inhibitor diisopropyl phosphofluoridate
and so this implied that Serene 195 must be the one that plays a major catalytic role in the active side of
that enzyme and so that's how we know that cine is the one that is involved in actually catalyzing the cleavage of the
peptide bonds at these specific bulky hydrophobic amino acids now the next question is what is the general
description of the reaction mechanism that takes place inside the active sides well basically it's a two-step catalytic
mechanism and we'll discuss the steps involved in much more detail in the next several lectures in this lecture we're
simply going to paint a general description a general picture of what takes place inside the active site and
how seene is actually involved in breaking and catalyzing those uh peptide bonds so we see that experimental data
suggests that kyrion uses a two-step mechanism to actually hydrolyze peptide bonds now the way that scientist
actually studied the reaction mechanism of kyrion is by using a special type of substrate molecule that once we react
that substrate molecule with the active side the product that is produced gives off a color and so we can study the
absorption of light when that colored product is produced and that will give us information as to what happens in
that reaction when we use kimat Trion so once again to monitor the reaction progress we generally use a substrate
that changes color when it reacts with the active side so we have this special special substrate molecule that when
reacts with the Serene inside the active side it produces a product that has a certain color to it and we can essent
monitor the color change and the absorbance of light as we produce that particular product so if we take a look
at the following graph the y axis is the absorbance of light of that product produced when the substrate is catalyzed
by the active side and the xaxis is basically the reaction progress so at the zero point at Time Zero we basically
take our mixture of that special prod uh special substrate and we mix it in with our enzyme and now our reaction begins
and notice what kind of curve we actually produce so we see that initially we have a very steep slope a
very large slope and then the slope begins to level off and so here the rate of the reaction is low but here the rate
of the reaction is very fast now what exactly does that tell us about the reaction mechanism of kimat Tron well
what tells us is we have this initial fast Burst stage and that describes the first step of the reaction and the first
step of reaction the actual binding of the substrate onto that catalytic sering of the active side is a very quick step
but as soon as that step takes place we have a second step and the second step is a slow step and so what happens is we
have the initial very quick step taking place and then eventually when the steady state conditions are reached when
the intermediate concentration concentration doesn't change too much we see that the slope begins to decrease
and so we have this relatively slow process taking place and that's essentially a result of the second step
of the reaction being relatively slow so graphing the data that we obtain from experiment from results this shows us
that the fast initial burst of the color product followed by by a slowing down and that's basically a result of us
reaching the steady state condition and that second step being so much slower than that first step so to see exactly
what happens let's take a look at the following diagram so in this particular diagram we have the active side of the
kimot Trion and this is Serene 195 so let's suppose we add our chromogenic substrate chromogen genic
simply means when that subst reacts to produce a product that product will change color and we can monitor the
color change we can basically determine the absorbance of light as the reaction progresses so we have this chromogenic
substrate we have the active side and so what happens in the first step is we have an illation step taking place so an
AEL Group found on this chromogenic substrate so this per purple group basically attaches onto the oxygen and
so when that takes place this uh this blue section the amide product is basically released and it's this amide
thing that produces that colored product and so as this reaction takes place the illation reaction takes place very very
creates a color change and we can monitor that color change on the following graph now once this reaction
takes place this oxygen of the Seine in the active side is assulated so this entire group is attached onto the oxygen
as shown and this is the Cove valent bond that is temporarily formed remember Chim tripson uses Cove valent catalysis
and that means we form a temporary Cove valent Bond so this intermediate is known as the AIL enzyme intermediate and
in the next step we have the water molecule that joins in the active side and it basically hydes the cleavage of
the peptide bond So It ultimately breaks this Bond here and it forms a bond between the oxygen and this carbon and
so in the second step we we have the deool step so this AEL group is removed from that oxygen the H from the uh water
molecule is given to that oxygen on the cine and so ultimately we regenerate that enzyme and then we also form that
hydrolized final product in which we now have a peptide bond that has been broken and so this is the very quick step that
takes place that accounts for this relatively steep slope but as we reach the steady state conditions as the
intermediate concentration basically does not change what begins to happen is this begins to level off and this
describes the fact that the Second Step takes place much slower than that initial step and so this is the twep
catalytic mechanism that basically generalizes what takes place inside kimat Trion so the first step is the
fast aculation step in which the AEL group is added onto the enzyme and then the amide is released and it's the amide
that is used to basically determine what the color change is we use it to basically determine how much light is
absorbed and then in the Second Step the second step is a much smaller step and in this step we basically break down the
AEL enzyme Intermediate by using water as a nucleophile and then we regenerate that enzyme and we form that hydrolized
final product now in the next several lectures we're going to look at a much detailed discussion as to what actually