Understanding Penicillin: The Mechanism of a Suicide Inhibitor

in our discussion on irreversible Inhibitors we briefly mentioned penicillin and we said that penicillin

is an example of a suicide inhibitor it's basically a molecule that binds onto an enzyme found on bacterial cells

and inhibits the activity of that enzyme it prevents the enzyme from actually forming the peptidoglycan cell wall

found around the membranes of bacterial cells now recall from biology that pep the gly and cell walls basically give

that bacterial cell the ability to resist a high osmotic pressure and it's because of that pep glycin cell wall

that once the bacterial cells enter our body that they don't actually immediately lies so once again that

Pepto glycin cell wall that we're going to discuss in just a moment basically allows that cell to resist a high

osmotic pressure now what we want to focus on in this lecture is the structure of

penicillin and the actual mechanism by which penicillin acts on that bacterial enzymes so let's begin with the

structure of penicillin so penicillin consists of three important components component number one is the five

membered ring we call thool Adine so thadine consists of this five member ring here now we also have a four member

ring here that's called the betal lactum and it's the instability and the high energy of the beta lactum that actually

gives that penicillin a reactive nature and allows it to actually react in the first place in fact it's this red carbon

in the betal lactum ring that allows it to react as we'll see towards the end of the lecture and finally the final

component of penicillin is this R Group so it's the R Group it's the variable R group that basically gives that

penicillin its unique structure so penicillin is actually a group of many related molecules that all contain the

thoolen ring the beta lactum ring as well as that unique variable R Group now how exactly did we uh discover the fact

that penicillin actually acts indirectly on the pep the glyc cell wall well in the late 1950s scientists carry out

carried out the following experiment they basically took bacterial cells they placed them into a hypertonic

environment and then they added penicillin and what they found was even though they had even though we had

penicillin those cells did not actually lie they did not actually die so if we took bacterial cells and grew them in

the presence of penicil in a hyperonic solution those cells did not actually die off so what do we mean by hypertonic

environment so on the outside of the cell we have a high concentration of solute on the inside we have a low

concentration so what that means is water is going to flow from the inside to the outside and so on the inside of

the cell we're going to have a low osmotic pressure now when the scientist took those cells and plac them into a

medium that we would typically find inside our body what they found was water basically rushed into the cell

that enlarged the cell and the cell eventually liced now they knew that if the cell contained pep the gly and cell

wall it would not lice and so what that means is that penicillin molecules somehow acted on that pep glycan cell

wall so again this experiment basically led to the conclusion that penicillin prevents the formation of the

peptidoglycan cell wall because it's the pep glycin cell wall that gives the cell stability structure and gives it the

ability to actually withstand or resist a high osmotic pressure that can exist once the cells are placed into the

environment that is found inside our body now the next question is how exactly does penicillin act on these

pept glycin cell walls well before we answer that question let's discuss the structure of pepid gly and then let's

discuss how the bacterial cells actually build this pepid glycin cell wall so if we examine this specific

strain of bacterial cells this is the type of Pepto glycan structure that we're going to see so basically pep

glycin means we have a peptide component and we have a glycogen component and so these purple molecules are sugar

molecules and they basically line up to form a linear polysaccharide chain and we have one chain we have two chain a

third chain a fourth chain and so forth and some of these sugar components basically are attached to short peptides

so we have this 1 2 3 4 amino acid tetrapeptide and we also have these 1 2 3 4 five so senta peptides and we see

that these tetrapeptides are connected to the tetrapeptides of the adjacent linear polysaccharide Chain by these

pentapeptides and these linkages are known as crosslinkages and they're the green linkages shown in these in this

diagram so the pep the glycan cell wall consists of long sugar chains that are connected by cross linkages between the

short peptides so this blue peptide here is connected to this blue peptide of the Json polysaccharide chain via this five

peptide so the pentapeptide short uh protein component now the question is what exactly is the reaction by which

the bacterial cells actually build these cross linkages in the first place well this is the reaction that is described

on the board so we have the pentapeptide so let's say the pentaglycine so this is glycine one glycine 2 glycine 3 glycine

4 and the terminal glycine that we basically Drew out now it's the nitrogen of this pentaglycine so this is the

pentaglycine shown here it's this nitrogen that's going to form a bond with this carbon here so this is an

adjacent short peptide and the terminal amino acid is the Dal alanine and the next one in line is also the Dal alanine

and what this nitrogen does is it bonds with this carbon and once it forms a bond it basically kicks off that

terminal deanine and that's exactly what forms this Green Cross linkage that we basically show here so this connects to

this carbon that kicks off this Terminal D alanine and that forms the Green Cross linkage between this terminal glycine on

the pentaglycine molecule and this deanine the second carbon uh the second deanine the carbon of that second

deanine so this is the reaction that takes place that forms the cross linkages in inside bacterial cells and

the enzyme that catalyzes speeds of this particular reaction inside bacterial cells is known as glycopeptide

transpeptidase so in bacterial cells this cross linking is catalyzed by an enzyme we call

glycopeptide transpeptidase and if we examine the active side of this enzyme the catalytic

amino acid the catalytic residue that catalyzes this reaction is seene so let's focus on how this

actually takes place inside the active side of this transpeptidase enzyme so this is a portion of the active side and

this is that catalytic residue that seing molecule so what happens is we take this blue molecule that we have

here that is basically attached to let's say the linear polysaccharide chain here and what happens is this seran molecule

the oxygen basically bonds onto this carbon and that kicks off this first DAL alanine to basically form the AEL

intermediate as well as this individual deanine that was removed and now what happened is we basically prepare this

section here so this molecule here to form that uh uh that cross linkage between this carbon here and this

nitrogen here so in the next step this pentaglycine would basically move into the active side and the enzyme would

catalyze the formation of that cross linkage between this nitrogen here and this carbon here so this is how that

catalyzation process actually takes place normally in the absence of penicillin now what happens when we add

ped pin well as we discussed in our discussion on irreversible Inhibitors we said that penicillin is an example of a

suicide inhibitor and what that basically means is what penicillin does is it moves into the active side of this

enzyme the glycopeptide transpeptidase and it tricks the active side the enzyme it tricks it into

thinking that the penicillin is actually a substrate and so this enzyme is going to treat the penicillin as if it was a

substrate it's going to begin the catalyzation process but as soon as it begins the catalyzation process what

happens is because of the reactive nature of the penicillin so remember from organic chemistry whenever we have

a structure a ring that consists of four atoms so four member ring that creates a lot of steric hindrance and that

increases the energy of that ring and it makes it very very reactive in fact it that's precisely why this carbon will be

very reactive and what will happen is when penicillin actually enters the active side of that glycopep uh

glycopeptide transpeptidase this seene will react the oxygen will react with the carbon and

that will break this Bond and that will relieve some of that steric hindrance that existed as a result of this high

reactivity of this uh a four member ring so the beta lacton and so once this bond is broken those two electrons in that

Bond will grab an H atom and will basically form this intermediate structure and as soon as we form this

intermediate structure that searing residue is no longer able ble to actually further catalyze that reaction

because we now block and inhibit that activity of that sering residue and so this is a suicide inhibitor because it

essentially tricks that active side into thinking it's a substrate it begins the catalysis process but somewhere down the

line it forms an intermediate that inhibits the activity of that transpeptidase and once it binds because

binds Cove valant and tightly it will not let go and so now that will inactivate the active side of the

glycopeptide transpeptidase and those crosslinkages will not be able to form and that will

basically break apart the structure of the pep glycin cell wall and the and the bacterial cell will now lack that pep

tolyan cell wall and this is precisely how penicillin actually acts so penicillin acts as a suicide inhibitor

it basically acts on that enzyme that is responsible for forming the cross linkages that hold these sugar

components inside the pepid the glycan cell wall of bacterial cells