Understanding Proteases: The Powerful Enzymes in Protein Hydrolysis

the enzymes of our body use a combination of different mechanisms to carry out their catalytic function to

speed up the rates of biological reactions and the four major mechanisms of action that we spoke of previously

include covalent catalysis acid-base catalysis metal ion catalysis and catalysis by proximity and orientation

and so to demonstrate these mechanisms we're going to begin our discussion on the first group of protein enzymes used

inside our body namely the protease so what exactly is a protease well a protease is a protein enzyme it's an

enzyme molecule that is a protein that catalyzes the hydrolysis the breaking of peptide bonds and peptide bonds also

known as amide bonds are the bonds that hold amino acids together in any protein molecule in any polypeptide chain now

why would we need to actually break a peptide bond in the first place well for instance if we ingest some food particle

that is a macro molecule for incident protein then we have to be able to break down that food protein molecule into its

individual constituent amino acids so that once we have those amino acids we can either use the amino acids to

actually form let's say ATP molecules or we can also use the amino acids to actually form brand new proteins and

brand new enzymes now the second reason as to why we need to be able to break a peptide bond is because our cells

actually need to be able to recycle protein molecules for instance if let's say a cell needs to decrease the number

of protein channels found in the cell membrane it has to be able to remove those protein channels and then digest

those protein channels and so inside the cells we have these digestive enzymes in the same way that we have digestive

enzymes inside our small intestine as well as inside our stomach and these digestive enzymes are proteins as they

are used to break down and recycle the proteins found inside ourselves and finally as we'll discuss in much more

detail in the future these proteases are also actually used in proteolytic cleavage and that is used to activate or

sometimes deactivate important biological pathways and biological molecules so as we'll see in the future

lecture these different types of digestive enzymes are actually themselves activated by other proteases

so the digestive enzymes inside our stomach for example aren't always functioning but as soon as we ingest

food those enzymes are activated by proteolytic cleavage by other protease molecules so these are the three major

reasons as to why we have to be able to break a peptide bond now the next question is why do we have to use a

catalyst why do we need to use an enzyme for this reaction to actually take place inside our body well as it turns out as

we'll see in just a moment the rate at which the reaction takes place is very very low so let's take a look at this

particular reaction so we have the peptide bond between the carbon and nitrogen shown in purple and what this

describes is the hydrolysis of the peptide bond so ultimately the water molecule acts as a nucleophile this

carbon acts as an electrophile and what happens is this nucleophilically attacks the carbon and ultimately displaces that

amide bond the peptide bond and notice that the arrow pointing this way is longer than the arrow pointing in

Reverse and what that means is equilibrium will lie towards the product side and that implies that the products

are lower in energy and more stable than the reactants so we see that even though this reaction is thermodynamically

favorable it doesn't take place at a very high-rate without the use of the

protease enzyme now why doesn't it take place at a very high rate well as it turns out water by itself is not a

strong enough nucleophile to actually attack the carbon and the carbon is not a strong enough electrophile and this

has to do with the strength of this amide bond so as it turns out this bond here shown as a single bond is not

actually a single bond this peptide bond contains a double bond nature double bond character as can be seen by drawing

these two Lewis dot structures so this is the first lewis dot structure but the other Lewis dot structure is described

by this diagram and so if these two electrons essentially go on to form a PI bond so let's use blue so we have these

two electrons basically form this PI bond here we get the following diagram and so these two electrons in this pi

bond between the carbon oxygen basically go on on to the orbital around the oxygen and we form a negative charge on

the oxygen a positive charge on a nitrogen but at the same time we have a double bond between the carbon and that

nitrogen so we see that although this bond isn't exactly a double bond it's also not exactly a single bond it's

somewhere in between because these two resonance stabilized structure describes the actual structure of that peptide

bond which is somewhere in between so because we have a greater electron density that is fluctuating in between

the carbon and nitrogen we have more electrons fluctuating between those two atoms the electrons of the oxygen will

not be able to get to that carbon because of electron-electron repulsion and that's exactly what we mean by the

carbon simply will not be a good enough electrophile and this oxygen on the water will not will not be a

good enough nucleophile for this reaction to take place at a high enough rate even though these products are more

stable and lower in energy than these reactants so we see that although this reaction is thermodynamically favorable

it occurs at an extremely slow rate and this has to do with the double bond character of peptide bonds in this

diagram we see that the resonance stabilized structure of peptide bonds make the carbon this carbon here less

susceptible to nucleophilic attack by water because of this resonance stabilization the electrons fluctuate

around the carbon and nitrogen as a result of the double bond character and that essentially electro electrostatic

repels the electrons of that water molecule and therefore in order for this reaction to actually take place at a

high enough rate inside our body and in order for us to be able to quickly and effectively break down these peptide

bonds we have to use these enzymes these proteases protease is as we'll see in the next several lectures actually make

water a much better nucleophile and they make the carbon a much better electrophile they make these reactants

much more reactive and that facilitates this hydrolysis reaction now we can actually categorize protease 'iz in two

different categories and these are five categories of proteases we have serine proteases we have cysteine proteases we

have metalloproteases and we have aspartate protease is and we'll discuss these in much more detail in the next

several lectures and we also have three onion proteases so let's very quickly discuss these four of the five protease

molecule so let's begin with serine proteases and by the way the major difference between these different

proteases is the presence of a specific type of residue inside the active side of that enzyme so in the

case of serine protease from the name you might guess that inside that active side it's a serine molecule a serine

amino acid that plays the nucleophilic role of nucleophilically attacking or breaking that peptide bond and so it's

the serine that ultimately catalyzes that reaction now in addition to that serine as we'll see in the next lecture

there are other additional residues present in the active site that also assist in the catalysis process as we'll

discuss in the next lecture now what are some examples of senior proteases and what are some of their roles so let's

begin with some digestive enzymes so we have trips and we have chymotrypsin we also have elastase and these three are

digestive enzymes found inside our small intestine which basically play the role of breaking down the proteins that we

ingest we also have seen proteases involved in the blood coagulation process and we'll discuss these in much

more detail when we'll discuss the blood cascade and so these are these are thrombin and plasmon now inside our

immune system we have the complement system and one important serum protease part of the complement system is known

as the complement c1 and finally see your protease is also play a role in reproduction so when we discuss sperm

cells we said that on the tip of sperm cells are these structures we call acrosome and inside the acrosome

are digestive enzymes and these digestive enzymes are known as acrosomal protease --is and these are examples on

serine protease so serine proteases are involved in biological processes such as digestion so trypsin chymotrypsin

elastase blood coagulation thrombin and plasmid we have immunity so the complement c1 and we also have

reproduction namely acrosomal protease which are the enzyme which are needed to basically digest the

whole inside the membrane covering of that excel so the sperm cell can move inside that excel to form that zygote

now we also have not only see our protease as we have cysteine proteases as Purtill or aspartate proteases and

metalloproteases so as you might as you might infer from the title cysteine protease is basically contain a cysteine

residue that plays that nucleophilic role of attacking that peptide bond and catalyzing this hydrolysis reaction out

cysteine proteases such as caspase and cathepsin are involved in programmed cell death also known as a pitocin and

this is basically an immune response that our body has and this process is also involved in a normal embryo logical

development of that human embryo now other evidence also suggests that we have cysteine proteases that are

involved in bone remodeling as well as MHC class 2 processing and remember MHC class 2 is a protein complex found on

certain cells immune cells of our body where MHC stands for the major histocompatibility class two complex

now these cysteine proteases are also found in many other organisms and they're found predominantly in fruits

and so papaya type of fruit contains a special cysteine protease known as papain now let's move on to as Purtill

or aspartate proteases as well as metalloproteases so once again from the title from that name you can infer that

instead of having serine or cysteine inside these active sites of these enzymes we have aspartic acid in fact

these enzymes contain two so a pair of aspartic acids and as we'll discuss in a future lecture one of those residues

takes away an age Adam and the other residue basically is used as a is used to increase the

nucleophilic character of that particular substrate molecule and Rina nor Renan is basically an example of an

aspir tool protease that is involved in increasing or decreasing so regulating the blood pressure inside our body and

we also have another example namely pepsin and pepsin is once again an example of a digestive enzyme it's used

to break down the proteins that we ingest into our body and finally we have metalloproteases and these are simply

enzymes proteases that actually utilize a metal atom a metal ion to basically catalyze that hydrolysis reaction and

two examples of such metallic proteases are carboxypeptidase a which is an example of a digestive enzyme we'll

focus on it in much more detail in future lecture and we also have a bacterial enzyme known as thermal Eisen

so we don't have inside our body but certain bacterial cells have the thermal icing protease molecule that is used to

break down peptide bonds inside that bacterial cell