lecture 13/4

enzyme mechansim -

how enzyme catalyz chemical reacitona dn were going to look at protease and an exmaple. protease are sometimes often refered to as peptidase. so thsoe two termas are used interhcnagely. prtease are a type of hydrlase. and hydrolase are enzyme that break chemcial bonds by addinga. water molecule. protease ar eta type that belong to the family and play many critial roles biological roles. inclduing development differentation cell migration immunity wound healing cell death and last but not least digestion.

if u eat chicken or steak or any kind of protein ur body cannot derive atp from it until u first break it down into indivual amino acids. and that process is shown . u conesume ur ptoeins as ur meal and then u have digestive enzyme which ar eptease that will break it down into smaller and smaller fragemtns until u ahve amino acids ur nody donest use all amino acids as a source of calories. but nowadays we usually intake enough calories that ur body doenst have to use all the amino acids to derive ernegy . instea a lot of it iused to syntheeize new protiens.

ribsoeomes they read the finroamtion in the gene but they need indivual amino acids are building blocks. there are two types of peptidase , endopeptides vs expeotide. endopeptidase will break peptide bonds in the middle of a protein of in the middle of a peptide. expopeptidase on the other hand will only cleave peptid ebonds at the very ends of proteins and peptides. there are two types of enodpeptide. the first one is calle amino peptidase oer aminoendopeptidase. those will break pepetie bonds at the ened terminud of the peptide or perotein. and then we have counter part the carboxypeptide which will cleave peptide bonds a the other end of the protein or pepetide. tehre are four main types of prtoease in our boyd. there classified according to the catalytic mechansim.

A is the active site of a serine proteainse B is the active site of a cystein protease. C is the aspartyle protease and D is the metalloprotease that uses zinc. they all break pepetide bonds. how they do it is different. so in the case of serine and cystein protease . its the serina R group and the cysten R group that actually do the cleaveage. wherease in the aspartyl protease and metalloprotease. its actauly a water molecule that does the peptide bonds cleaveg and the cataltyci resuides are there to simpley active that water mocluel,. so were going to look at the serine protease in detail today and see how they accomplish their job. there are many proteases in our boyd.

their estimate is about 2% of our genes encoded for protease. and that tells u that its very improtnat class of enzymes. so here are just three examples of serine protease thats found inr our body. trypsine chymotrypsine and elastase. these are all digestive enzynmes. they are going to help digest food. elastase is a digestive enzyme but also helps for our immune funciton it has a dual role.

u can see that the structure is verys imilar. they all utaulize serine residue to hydrolyze pepetide bonds and they all contain iportnat serine at the active site. but its not just serine. they all contain whats called a catalytic traid that made up of serine, hisitinde and asparatte. and all three are very importnmatn. although its at the endo f the day the serine that does the cleaveage. exame two remember the amino acids, structure name three letter code and one letter code. proteases are very importnatn but there is a problems, just like any other protein its syntehsized by the ribsoeome in the cytoolasm of your cells.. 9

this could cause havoc i fthe protease starts breaking peptide bonds of various proteins thats inside the cell. so how does nature solve this probelms. when trypsin is newly synthesized. this is not ht eprotein that ribsoosme synthesizes. actaully what the ribsoeoms syntheisze is called trypsinogen. this has two additniaotnal amino acids at the N temrienus. so its longer than trypsine buyt just two amino acids but those two amino acids cause this protein to have a different shape. and in this shape the active site reside are not in the correct psotiion and therefore trypsinogen are catalytically inactive this is very importnat beucase u dont want these digestive enzymes to be active in ur helathy cells. its going to start digesting all the proteins. that u really ned. so trypsin , trypsinogen is syntehized and then its exported and its evnautally targeted to the smalel intesine cativity where its needed. thats where ur protein gets digested, there there is a endopeptidase that will cleave off the two additonal amino acids. once that cleaved and gone itll change its conformation to this shape and now that the native ocnromtion of tyrpsin and that enzyme is not fully active and it can go about digesting ur meal. same stragety applies to chymotrypsin and elastase. its intaily synteheiszed in an inactive forma nd it gest sactived only once it reaches it s final destination. for trypsin and chymotrupsine thats ur small intesitine.

look at chymotrypsin, its an endopeptidase. it breaks peptide bonds. so thsi enzyme has two substrates. ut has the peptide or the protein which is going to bind to chmotrypsin and break the peptide bond. it has a second substrate though and thsi is water. water here is very improtnat without water ur not going ot accomplish or chymotrypsine is not going to accomplish its job. most enzymes ar speicif as to which substrate it acts on. so we say bc of this enzymes have a high susbtrate specificity. its not just going to bind any substrate, thats bs the active site shape dicstates what is binds and what it doesnt bind. proteases have a relatively loose substrate specificity that means its going to bind many. different proteins and peptides. and this is unsual in the enzyme word. u cant have protease for every different peptde bond youre going to cleave. so chymotrypsin will act on many different peptides and protiens however it has a preference. it prefereintially cleaves peptide bonds involving amino acids iwth bulk hydrophobic R groups like phetynlalalnine tyrosine and trypsin. it will act on other peptides and protiens but it will prefernetially cleaves the oens that. have these hydrophobic bulky R grups. tyrpsin is differne. it prefers bassic resudies . what are some basic residues. lysine arginine, elastase prefers small nature residues, liek alanine glycine. ,valine, serine. so among the three of them it can cleave any peptide bond thats found in your meal. the execption though is gluten.

celical disease and gluten. 1 in 100 ppll get sick twhen they get gluten containg food. glutein is a protein and it turns out that non our of digestive enzymes are good at digesting gluten. chymotrpsin trypsin elastine none of those can break gluten peptide into indivaul amino acids. and fior most of us thats fine. u can use the toilet and that comes out of our system but in 1 in 100 ppl the undigested glutena ctivates our immune systema and starts attacking the epthital cells in ur small intesite and that causes problesm. so why is chymotrypisn trypicin and elaste not good at digetsting gluten? its because gluten is 50% proline. if u look at the primary structure , prolines are hard to digest. because it has the second covalent bond where the R group is bondedn to the nitrogen. here ius the cystal structure of chymotrypsin.

in cyan up there bound to a peptide which is colored and purple down there te catalytic traid serine histide asspartate. chymotrupsin is a serin protease because it has the critial serine residue that does the cleaving.

mecahsnim has seven steps. its not a ciruclar pathway. that means when everything is doen it goes right back up to the first step. youll encounter other circular pathways. some pathways are lienar like glycosis and some pathways are cylci liek citric acid cycle. this oen is a critic pathway bc chynotrypsin is a biological caytlsit. whatever it does it should not change. after its jobis done it needs to go back to its orignal state so that the cataylst can be reused to carry out another round of the raciton. so lets look at the chymotrypsine active site becauser anything happens.

step ONE.

so the white space is the active site. this is where eventaully the substrate will coem and bind four resides are highlighted here aspartate, histidine, serine, and glycine. however it does ot mean that only these four residues are present in the active site. the active site are lined by many many other amino acids. these four ar ethe ones that are direclty involes. the other ones are importnat as well. without them youre not going to form this improtnat active site cavity shape. these direclty importnat catalytic residues will not be oritented corretly if it wasnt for all the neighboring amino acids. withing the active site there are towo locations. one is called the hydrophobic pocket which is the. yellow bubbcle and the other one is the oxyanion hole . theyll beocme very improtnatn. first step is simply binding o f the peptide substrate. So E plus S going to ES.

so there is

so what is drawn here is that there are three amino acids, amino acid one in red , two is in blakc and three is in blue. so u know chymotruspin can accept or bind tripeptide . or it can be much larger peptide. but the portion that binds at the actie site would be a three residue secion of that parent molecule. so u can think of it either way. just for simplicyt were going to treat this as a short tripeptide. but dont forget it can be a sectio of a larger [rptein . so when chymotrupsin finishes its job. itsgoing to break the peptide bond between C and nitrogen coavlent bond between the blakc and blue amio acid. so the product will be a dipeptide consistiof o the red and black amino aic. the water is a subtrate but it will et consumed. how it binds ot the active site. intially it will bind by non covalent interacitons the first one is the H bond between the backbone of the serin to the carboxyl oxygen of ur peptide. the peptide in the a ctive site.

the second one is a hydrophobic interaction btween the R group of the black amino acid and the hydrophobic pocket. . its another noo covalnet interaction.

now u can see why chrymostrupin prefers to cleave peptid bonds associated with amino acids with a hydrophobic R group bulk R group. thats because thsi itneraction this hydrophobic interaction is maximized. so in this example u have a phenylalaine there. whic is perfect fit for the hydrophobic pocket. if u have something else like aspartate instead of phentylanein its sill going to bind but its going to bid more weakly. so thats why we say this enzyme has prefernce for amino acids iwth a hydrophobic R group.

so step two. that histidien R group , remember histiine R groups is oe of thsoe that can be protoanted or dpeorontated, it starts in the deproaotned fomr, so hsitine R group is acting as a base and its abstrating the hydrogen from the hydroxyl group of that serin. and once u deproatnate tha hydroyl group u have an alkoxide which is going to act as a nucelpthile and attack that carboynl carbon atom of the carboynl group of the peptide bond that were trying to break. before the nucelphilic attack happens u know that carbon has a planar gemotry. but once that ncueltphilic attacks what u form here, u have a tetrahedral carbon atom because now its attached to four dif groups. ur substrate is covetnly bound to the enzyme by the cabron. o2 bond. the o2. ispart of hte protein its from the serin R group. and that carbonis part of the peptide susbtrate and now u ahve a covalent bond. u form an acyl enzyme. this is rare . its one of the stragetigeies wher eu forma. covalnet bond between substrate and enzyme. u forma. atetrahedral intermdiate but hte

oxygen is an oxyanion meaning it has an unpair elecotnr where the negative charge comes from an oxyanions are hihgly hihgly unstable. however when the oxyanion is formed inside the active site is stablized by these hydrogen bond donor groups taht near the oxyanio hole.

that oxyanion is stablized, serina nd glyine is going to promoote formation of the oxyanion. however even if its stablized by those hydrogen bonds, its hihgly reactive and the electron pari will collpase back and reform the carboyl group as before. but the problem is at this stage u alrewad have four covalent bonds . on that cabron atom. and u know that this ist he max number of covaletn bonds that carbon can form so something else has to give and that is that cabron nitrogen bond. that will break as that new double bond form.s. and taht nitrogen at the same time will grab the hyrogen from that histidine and now this blye amino acid is not bound to anything. by covalent bond or non covalent interaction . now that the blue amino acid can diffuse out of the active site and what ur left with is just the dipeptide portion thats covalently attached to ur chymotrupin. this was step three

the mission is compelted the peptide bond was broken. but its not compeltely because the enzyem is not back to its og conformation. if we just elave it at this it cannot be resued. s

so step four. when water binds. that site wehre the blue amino acid use to be is where a water mocluel is going ot ocme in and bind and its going ot hydrogen bond to that histine reisude and youre going to formt his compex.

step five is wehre the histine is going to act as a base and asbstrat the H form the water mocleula dn from a hydroxide and now this hydoxide ion thats going to do the nucleptphilic attach same thing as before youre going to in the process youre going to form a oxyanion thats sort of okay because u have the hydrogen bond the donotrs that stiabilziing that ot an extent. and in the next stpe youre going to refrom that carbonyl bond as we did before.

in the next step youre going to reform that carbonyl bond as we did before and ur also groing to break that usbtrate to enzyem covalent bond. so that dipeptid protion is not covaltnely linked to the enzyme and step 7 is simpley the depatrutre of taht sefcond product mocleule which is the dipeptide. and if u look at the active site were right back to where we started with. and the job is done. this can accept another peptide and go through this whole process again and again. the very defintinao of an enzyme by the way so this has seven steps . which one is the rate limitng . for chymotrupin depeonds on what the substrate peptide is but in most cases its step five wher ewater attcks thsi is si a slow step because a hydroxide ion is not a good nuceltphile. and thsi si relatevely slow so this step numbre five pretyy much dictatse how fast the overall reaction will go.

just review so chymotrupsin is a serin prtease , it has a serin residue that acts as a nuceltphile but its not the only residue that simprotnat it has thos e serine histitne and asparate triad thats all incolved. so also that glycine to a lesser extent but we dont include that in the traid that glycine that that donates a hydrogen bond.

so studying protease are very imprtonat one example has to do with HIV. its a retroviruse so it has an RNA genome. it uses reverse tranciptase to convert RNA to DNA and then it uses our own cellular machinery to tranlate the gene to protiens. so wahts unique about how this virus makes protein is that HIV viral genes are tranalted into large polyprotiens, its like a bunch of different proitnes all covalently linked togheter. and after that trnaltion is compelted it uses special protease HIV protease to break those covlaent bonds and serpate them into indivual protiens. we dont do this but viruses do . and this is importnat because if we can develpo an inhibtiro against the HIV protease we should be able to stop HIV from replicaitng okay so this is the active site of HIV protease its not a serin protease. its a sparyatle protease thats the catalyticaly active residue and actally its not the asparte asparatae doesnt act here as the nucletphile instead it acts, it activates the water molecule. it abstracts that hydrogen ion and its the hydroxide that is the nuclephole. so hers an example of a aspartyl protease. and we have good HIV druggs now. So HIV affected indivauls. can live almost a full life. and thos inhibitors are substrate analogs. so heres a peptide that usually binds ot hte HIV protease. so this is the natual peptide for that protease. heres wher ethe HIV protease will cleave. but if wel look at drugs like this squanvari and darunavir. from far away they will look like peptides. so sceintsit designed these inhibotr molcuesl and they did that by micmking the natural substrate peptide. but if u look at them in detial its nota. antural peptide and therefore once these molcuels bind ot hte active site they do not get cleaves they just say bound. effecitly stopping the protese from working. many of these are actually non reveresible inhibors of the HIV protease. im gong to show u a series of three short vidoes.

watch the videos -

last part indiuce fit, most enzyme use the induce fit mecahnsim. there is an enzuyme called hexokinase the substrate is fglucose 6- phpphoate so teh substrate is gluce and atp, product is glucose 6-phosphate and adp. and when they

xample, there's an enzyme called hexokinase that

catalyzes phosphorylation of glucose to form

glucose 6-phosphate. So the substrate is glucose

and ATP, product is glucose 6-phosphate and ADP.

Now when they solve the structure of the ligand

-free enzyme. That's what it looks like. And at

the bottom, this is the crystal structure of

the same enzyme that has ADP and glucose bound,

okay? And I don't know if you can tell very easily,

but the shape is actually quite different, okay? So

this is not lock and key it means that as the substrate

binds to the enzyme the shape of the enzyme changes

to accommodate that substrate okay and to optimize

the the arrangement of the active site residues okay

the chymotrypsin we talked about today also uses induced

fit mechanism so the structure of the active site

okay even though in the cartoon diagram they drew it

exactly the same, whether the active site is empty or

substrate is bound, in reality if you look at high

resolution crystal structures it's different, it's

slightly different and that's important for its catalytic

activity.