enzyme catalytic mechanisms...oneby :- hanan jamal and rand khlaifat page 1 enzyme catalytic...

Enzyme Catalytic

Mechanisms الفريق الطبي األكاديمي

كلــية الطب البشري

البلقاء التطبيقية / المركز

6102/6166أحياها و من

DoneBy :- Hanan Jamal and Rand Khlaifat

Enzyme Catalytic Mechanisms و من أحياها

6102/6166

As you see , you see this polypeptide chain and it's N terminal is Glycin

and the C-terminalis Asn (Aspargine), up on you subject this

olegopeptide to protease say Seryin protease , Ser will bind to the R-

group of phenylalanine as it mention last time this is the C-carboxylic

group of this phenylalanine , so it will cut or break this bond as a result

you will have this phenylalanine attach to the polypeptide chain + the

rest of polypeptide chain.


6102/6166

*What type of bonds that will be broken by chymotrypsin ?

Peptide bonds-

How peptide bond will be broken by chymotrypsin ? *

-It was found after many experiment that chymotrypsin did it's action in

two phases:

1-vary fast phase (Burst phase). 2-slow phase.

So you are going to look in details at the active site of chymotrypsin and

see going on what is about the Burst phase (fast) and slow phase of the

reaction.

So you see A product is form in a Burst phase and also more product will

be produced slowly in the slow phase .


6102/6166

Now this is the substrate of chymotrypsin this are represent the rest the

phenylalanine R-group as well as the rest of polypeptide chain and the

line represent the other things of the polypeptide chain, but what is

important to us here is this bond which is peptide bond and this are

which is R-group of phenylalanine .

What is the R-group of phenylalanine ? *

-Aromatic ring or benzin which is a hydrophobic and it will be bound to

the enzyme.

Now this is represent a very close look at the active site of chymotrypsin

and as you see Ser , His and Asp and they are called Triad catalytic side.

This triad is composed of Ser, His, Asp.

Now remember these 3 amino acid 8;02 which is constitute the active

site of the enzyme , they are far from each other in the primary structure

, they are not close to each other , but what brought them close here in

the active site?


6102/6166

Tertiary structure or foldin of chymotrypsin that brought these amino

acid (triad) together in order to catalysis , the break down peptide bond.

ll as His and (9;23) as weamineNow this Ser is very important as well see

less important of Asp.

What is enzyme back bone ?

The rest of amino acid that constitute the enzyme , we just look at active

site which is the rest of the peptide bonds as well as the R-group of the

rest of the amino acid ,

What is the number of amino acid in the chymotrypsin ?

3 amino acid, but not only 3 maybe one hundred but we are

concernedonly now in the triad (active site).

The backbone is the remaining of 3 amino acid.

Now concentrate on this on which is the R-group of the Ser , and this

imidazole which is the R-group of His and this carboxylic group of Asp.

This is what happened when the substrate binds, what do you think

when a substrate bind will happen to the enzyme or active site ?

Structural change , very very small structure change , this is very small

structure change brings the triad much close to each other in what is

called close proximity will oriented to each other in order to start

chemical catalysis.

So what you see here is the the R-group is bound to the substrate

binding site of the chymotrypsin and the substrate binding site of the

chymotrypsin is called S1 pocket in which it will bind the R-group of the

phynelalanine or tyrosin or tryptophane , only sometimes it could bind

big hydrophobic non polar R-group like( 14;05) , but in most of the times

it binds the R-group of aromatic amino acid , phe , ter, tryp


6102/6166

The S1 pocket must be non-polar in nature in order to bind the R-group

of aromatic amino acid .

Now when these amino acid in the triad become very close to each

other , this is negatively charge and full of electron and here also it is rich

in electron , so when they are come in very close to each other all

electrons in the imidizole will sink in this region they will accumulate in

this region.


6102/6166

Serine Proteases• Catalytic Mechanism

1 . Binding of Substrate Stimulates

Slight Structural Changes

2 . Structural Changes Induced by Binding

Change Electronic Environment of Catalytic Triad

Now this H positively charge and here we have a lot of electrons , so

those electrons will pull this positively charge proton and leave this

oxygen atom in the Ser, so what happen , then H+ will be pulled to this N

because it's rich in electrons and this H is positively charge at the same

time this H will be pulled to those electron on oxygen , and this is a

product , what do you see here , the H on OH has been left , pull to the N

of imidizole and this H will pulled to the oxygen , this O- on the R-group

of Ser is called the alkoxide ion , this is very reactive species of oxygen so

when takes this H from Ser , leaving O as negatively charge and alkoxide

ion very highly reactive and it will attack this bond which is the bond of

substrate .

The peptide bond of the substrate , so it will attack it and break it, so

because of the alkoxide ion is formed and it's highly reactive it will

attack the peptide bond and break it as you see here.


6102/6166


6102/6166

Now when attack to it , it will form a non-stable intermediate called

carbon tetrahedral , this is the carbon tetrahedral which is intermediate

and not stable , now this non stable intermediate must be protected in a

region in the enzyme called Oxyanion Hole , if it's not protected by

oxyanion hole it will form covalent bond with the enzyme and kill the

enzyme.

And as a result of this breaking , you will break the peptide bond and you

will end up with part of the polypeptide chain covalently attach to Ser

and second half is released , this released is represented by the Burst or

fast stage that you saw in the curve at the beginning , so the released of

half of the peptide bond as you see here as a result of the oxyanion hole

and alkoxide attach it breaks the peptide bond into two halfs:

1-that has the N-terminal half

2-that has the carboxily group half and it's covelantly transetionaly

attached to the enzyme Ver, Ser and the N-terminal half is released.

The released of the N-terminal half represent the Burst phase of the

reaction.

The slow phase in which water is participating in the reaction , now

remember that proteases are hydrolyses means break bond in the

present of water.


6102/6166


6102/6166

What happens when water enters the active site?

The nitrogen atom of imidazol ring ,which is rich in electron, pulls the

hydrogen atom of water to it. Once hydrogen is pulled, it will leave the

hdydroxyl group ‘OH’ as free radical. As a result, the oxygen of water will

attack the carbonyl carbon of Ser and breaks the the bond – the second

half- .

Tetrahedral intermediate forms again, and it must be protected in the

oxyanian Hole. Then the oxygen atom of the tetrahedral abstracts the

Hydrogen atom on N-Group to reform(return) the hydroxyl group of

Serine, and the rest of tetrahedral is released.


6102/6166


6102/6166

The second half of the peptide is released. So, the bond will be broken,

the Serine comes back as OH and everything comes back as it started.

*The second phase is very slow; because until water reaches the

reaction it will take time in order to break the other half of peptide

bond.

*What was the role of Aspartic Acid in the reaction?

It pulls the hydrogen atom from nitrogen of imidazole ring to create the

electron sink in the imidazole ( it is a minor role, but it has an

importance on the catalytic triad).


6102/6166


6102/6166

Not all proteases have the same S1 pocket. For example, Aspartate

proteases have different S1 pocket from Serine proteases and Histidine

proteases too.

S1 pocket nature of chymotrypsin must be nonpolar (hydrophobic)

Examples of hydrophobic R groups present in S1 pocket of chymotrypsin:

Glysine, Alanine, Valine, Leucine & Isoleucine.

These reagents are very important in determining the primary structure

(the sequence of amino acids) of a protein.

*Trypsin hydrolyses peptide bonds after (basic) amino acids in the

polypeptide chain, like Lysine and Arginine. The nature of S1 pocket of

trypsin is acidic (negatively charged)- rich in Glutamic acid and Aspartic

acid- because Lysine and Arginine are positively charged. So, in order to


6102/6166

bind in complementary proper orientation, R groups in S1 pocket of

Trypsin must be negative in nature.

*Elastase is a protease that hydrolyses peptide bonds after Glycine,

Alanine & Valine. The nature of R groups in the active site of Elastase is

hydrophobic.

Scientists did an experiment in which they start mutating chymotrypsin

(changing amino acid residues in the active site one by one). This process

is called Site-directed mutagenesis and it means {changing of one amino

acid in the active site once a time then measure the activity of the

enzyme}. It explains how one amino acid could determine the

composition of the active site.

*Mutation of serine or histidine to alanine in the catalytic triad has as

negative effect on activity as mutating all three acids to alanine.

Mutating aspartic acid has less of a negative effect.


6102/6166

Serpins are very important; because in the body we can’t let the

protease to be active all the time. If it is active all the time, it will

hydrolyses the proteins of cells and tissues and kill them. So, our cells

have a system to control activity of proteases and the system is the

existence of inhibitors for those proteases.

Alpha-1-antitrypsin is an inhibitor for proteases (one of them elastase) in

the lungs. Elastase dose it work (killing viruses and bacteria that enter

our lungs) but when the cell does not want it to work in the lung, it

produces alpha-1-antitrypsin to attack and inactivate it.

In smokers alpha-1-antitrypsin is damaged; because of hydroxyl free

radicals that will oxidize the methionine residue which’s important in the

active site of alpha-1-antitrypsin and inhibit it. So, elastase will continue

damaging proteins in smoker’s lungs and resulting in Emphysema.


6102/6166

Note: Alpha-1-antitrypsin is named mistakenly; it is not only anti trypsin

but it is anti all serine proteases.

enzyme catalytic mechanisms...oneby :- hanan jamal and rand khlaifat page 1 enzyme catalytic...

Documents