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Biochemistry Elliot January 10, 2014 10:00pm-11:00am NT #22 Enzyme Regulation and Signal Transduction Slide #74: Clinical

correlations behin this! This is "in o# ne$! There are a number o# rare mutations that occur in that %&T 'ene! (here you 'et loss o# #unction an yiel s e)treme insulin resistance! (hat you en up $ith is a sin'le point mutation that chan'es this 'lutamic aci to a lysine! *lutamic aci is ne'ati+ely char'e , lysine is positi+ely char'e ! That,s a si'ni#icant alteration in a protein sur#ace! This mutation occurs on $hat,s calle the plekstrin bin in' omain! -lec"strin is an a aptor protein that allo$s t$o proteins to bin to'ether! The mutation o# plec"strin allo$s proteins to bin to it constantly! .# it,s boun to plec"strin constantly, it,s 'oin' to bin to the "inase an allo$ it to interact $ith GLUT4 transporters, an recruit them to the membrane constantly! /o you,re 'oin' to ha+e *01T4 transporters in muscle an a ipose in the membrane constantly! They,re not suppose to be there, there only suppose to be there in times o# plenty! /o muscle an a ipose ta"e up all the 'lucose in the bloo an lea+e little #or the brain, $hite bloo cells, an re bloo cells! /o *01T4 is there all the time! 2uscle is constantly ta"in' 'lucose out! /o you 'o to be an your bloo 'lucose $hile you,re #astin' comes o$n, an normally $hat happens is the li+er ma"es more 'lucose to stabili3e 'lucose le+els o+er the ni'ht! But $ith this mutation $ith *01T4 transporters constantly in the membrane, your 'lucose le+els $ill "eep 'oin' o$n an you mi'ht $a"e up in the mornin' $ith pro#oun hypo'lycemia! /o ho$ o you compensate #or that4 5ou can eat comple) corn starch be#ore be ! % +ery comple) 'lucose boun polymerically that bro"e o$n +ery slo$ly in the 'ut so that it continuously release 'lucose in the *. tract, $hich then 'ot into the circulatory system! This "eeps your 'lucose le+els hi'her throu'h the ni'ht! Slide 75 7!: Ne)t thin' $e,re tal"in' about is allosteric control! This is ho$ you control a comple) en3yme! (e are 'oin' to loo" at this control o# aspartate transcar"amoylase $hich produces cytidine trip#osp#ate $%T&'! .t is the rate limitin' step in this reaction! Slide 77: This is the crystalline structure o# the en3yme re uce to alpha helices an beta sheets! There are si) catalytic subunits, there are si) re'ulatory subunits! /o $hat happens here is $e,re only at physiolo'ical substrate concentration here! /o 2ichaelis-2enten 'i+es you the #alse sense o# security that you can 'o #rom small concentrations o# substrates to hu'e concentrations o# substrate in a li+in' cell! But you ha+e a +ery small $in o$ o# substrate concentration in a li+in' cell! /o the concentration o# aspartate is 'oin' to be in a narro$ ran'e! There#ore . ha+e to #i'ure out a $ay to re'ulate the en3yme so that it has a hi'her a##inity #or the substrate because . can,t 'et the cell to increase aspartate on its o$n! /o . ha+e to mess $ith the en3yme to impro+e its a##inity #or $hat substrate is there! %n that,s $hat this is about! 5ou 'o #rom this 2ichaelis cur+e to a si'moi al cur+e! 5ou start to bin substrate an then you can bin a $hole lot o# substrate! 5ou start to release pro uct, you release a $hole lot o# pro uct! Slide 7(: /o this is $hat the structure loo"s li"e! %n ho$ it $or"s is a monument to structural biolo'y! 1n er tense non-reacti+e catalytic con itions, theses si) catalytic subunits collapse

an lipases are ma e as pre-en3ymes or 3ymo'ens: inacti+e! Entero"inases in the 'ut linin' sees trypsinogens an clips it an the $hole system reor'ani3es into the acti+e #ormation! %t this point.it tells you t$o thin's! 8ne. let.s a lot o# purine nucleoti es.bin s. put in some CT. the acti+e trypsin sees other tar'et proteins li"e chymotrypsino'en. an 14: an 14> to #orm c#ymotrypsin! This is +ery common! Slide: (. ha+e plenty! There are also allosteric acti+ators! %T-! .. $ant to ma"e a lot o# pyrimi ines to balance it all o##! /o there. . it #orces this comple) open! /o it. $ante to sho$ you is that e+ery en3yme here is a serine protease. there.# . ha+e a lot o# %T-.an %Tare purines an you $ant about an e9ual amount o# pyrimi ines.m not ma"in' any more pro uct because . there. elastino'en! % lot o# these proen3ymes 'et clea+e at speci#ic bon s that allo$ them all to reor'ani3e an #orm the acti+e site! /o here is c#ymotrypsinogen an it is clea+e here bet$een resi ues 1< an 1=.then the substrates are bloc"e an .: &epsinogen is ma"e in the stomach linin'. 1. not substrate. T. put in some %T. because you. can rop the reaction rate to about :.s use it to buil somethin'! T$o.s in ynamic e9uilibrium so there is constant #ine tunin' 'oin' on! Slide #(* (+: 8" no$ .re usin' them to ma"e 6N% or 7N%.s an allosteric acti+ator! Slide #7): /o i# you put in a little %T-. it is aci sensiti+e so $hen it 'ets to a p? o# about t$o or three! 5ou protonate pepsino'en an it mo+es its o$n bloc"in' site into its acti+e site an cuts itsel#! Then it reor'ani3es to that it cut other aci enature ietary proteins! Slide (-: ?ere is the clottin' casca e! 5ou o not nee to "no$ the clottin' casca e ri'ht no$! But $hat . amylases. the pro uct o# this lon' path$ay comes bac" an bin s to an allosteric site! %n allosteric site is a speci#ic bin in' site o# CT. an $e $ant to ma"e about an e9ual molar amount o# pyrimi ine nucleoti es! /o *T. your 7N% an 6N% polymerases $or" more e##iciently! /o i# . an you nee to ma"e more o# this stu##! These sites are not 'oin' to be occupie at $hich point they chan'e con#ormation an the comple) e)pan s or opens! (hat.ll tal" about co+alent mo i#ication by proteolysis! 7i'esti+e proteases... increase the reaction rate about a 1000 #ol ! . i# you put a little CT.s a lot o# ener'y. but as a 3ymo'en or inacti+e! Slide (4 (5: %nother $ay o# re'ulatin' en3ymes is co+alent mo i#ication! 0et. it oes not occur on the catalytic subunit! But CT.! %'ain these thin's are not all on or all o##! .o$n on themsel+es! /o it.bin s to this allosteric bin in' site an chan'es the con#ormation o# the re'ulatory subunit $hich is connecte to the catalytic subunit an causes the entire comple) to collapse on itsel#! No$ i# CT.s another site on the re'ulatory omain that bin s %T-! (hen the %T.that occurs on the re'ulatory subunit.t 'et substrate into it. you acti+ate the system..s 'o bac" to "inases an $hat they o an ho$ they $or"! Glucagon is ma e by the pancreas $hen . you can.it eacti+ates the system! %n you $ant to loo" at this #i'ure here.# the acti+e site is bloc"e . no reaction.s 'oo about that is the acti+e sites are on the internal sur#ace! (hen they collapse o$n on themsel+es they literally collapse o$n so that the protein is bloc"in' its o$n acti+e site! .# there is a lot o# %T.t.s nice an pac"e ! The en pro uct. you can see i# . an reaction rates rop ramatically! /o i# . ha+e plenty o# CT.le+els ecrease. cyti ine triphosphate. an C! That $ay.

t. acti+ate one system an inacti+ate the other simultaneously! /o this is nicely re+ersible! There are t$o . phosphorylate it. you pump out more so ium an $ater into your intestinal tract! Slide #(7: -&% is actually a tetramer $ith t$o re'ulatory subunits! c%2. but its tar'et proteins reco'ni3e it an start a $hole bunch o# si'nalin' casca es +ery rapi ly! /o .n this case the si'nal trans uction protein is a G protein! * stan s #or GT& so this is a *T. ha+e a li'an that bin s to a receptor that acti+ates a trans uction protein that bin s to an en3yme that creates a pro uct that turns on a si'nalin' casca e! 7espite c%2.' 'ets acti+ate #or a lon'er perio o# time! .# more so ium lea+es the cell more chlori e lea+es the cell $hich causes more $ater to lea+e the cell! (hy o you thin" e)0a) has so much ca##eine in it4 . use one si'nal. put a phosphate on! .into c.t turns c%2. .ll see in the case #or insulin . inhibit phospho iesterase.bin in' protein! . $hich may in uce inappropriate metabolism or 'ene e)pression! %a00eine inhibits phospho iesterase! . phosphorylate these proteins.# you ha+e a lot o# ca##eine in your system. it has a protein that brea"s it o$n! &#osp#odiesterase! .# you i n.is hy roly3e to %2-. $hich in uces a chan'e in con#ormation o# the * protein #or a much hi'her a##inity #or *T-! /o *T.s a small polypepti e! This polypepti e has a really hi'h a##inity #or a receptor! Slide (!: /o 'luca'on @or epinephrineA bin s to its speci#ic receptor! The receptor chan'es con#ormation $hich is transmitte to a si'nal trans uction protein! .t phosphorylates its tar'ets #or a lon'er perio o# time! 8ne o# the tar'ets is a so ium transporter. phosphorylate a protein to acti+ate 'lyco'en brea" o$n.comes in an islo 'es the *7-! This is the acti+ate protein! This acti+ate * protein oc"s $ith a tar'et protein! . can acti+ate it $hen ./&! c%2. $&1. they e)ist! 5ou.to %2-! 5ou nee this because $hen you si'nal a system to o somethin'.bein' unstable.n this case the tar'et protein is adenylate cyclase! % enylate cyclase. the re'ulatory subunits re'ain their a##inity #or the catalytic subunits an bloc" the acti+e sites o# -&%! Slide #((: No$ actually there are a $hole lot o# "inase proteins! % "inase ta"es %T. but it can easily 'o the other $ay! .'lucose le+els are lo$! . c%2.# . an phosphorylate another protein to inacti+ate 'lycolysis! /o i# . cytosolic "inases! ?ere .t 'et ri o# this c%2. you $ant to pulse it! -ulsin' allo$s you to o #ine tunin' increments o# $hate+er metabolism you $ant to a Bust to accommo ate the en+ironment! . ha+e a protein that becomes inacti+e $hen .stays aroun a lot lon'er! /o this protein kinase .bin s to these an they lose a##inity #or the catalytic subunits $hich acti+ates -&%! (hen c%2. once it oc"s $ith a * protein is turne on an turns %T.n the inacti+e state the * protein is boun to *7-! 8nce the li'an bin s to the receptor.or *Tan phosphorylates other proteins! ?ere a "inase is acti+ate an attaches a phosphate to an alcohol 'roup! There are t$o classes! There are serine t#reonine kinases that stic" a phosphate on a serine or threonine an tyrosine "inases! The tyrosine kinase #amily typically ten to be membrane receptor proteins! The serineCthreonine "inases ten to by #ree-roamin'. the receptor chan'es its con#ormation. so more so ium lea+es the cell! .you $oul be recruitin' a $hole more o# these cyclic systems.is some$hat unstable an e'ra es.n #act.

acti+ates -&% an this oes a number o# thin's. -&% $ill acti+ate proteins #or 'ene e)pression so you can 'et lon' term recruitment o# en3ymes nee e to support 'luconeo'enesis! /o metabolically this system reacts 9uic"ly. $hich ma"es c%2-! c%2.nsulin also acts throu'h its o$n tyrosine "inase receptor an tar'ets a lot o# i##erent proteins! .t $ant to burn 'lucose $hen it is lo$! That.s missin' an en3yme! Glucose4!4p#osp#ate stays as 'lucose-=phosphate so it is trappe in the cell! 2uscle cells cannot ta"e that phosphate 'roup o## li"e li+er cells can! Slide #)+ ). li"e ten#ol slo$er! This allo$s the phosphorylate tar'ets to e)ist #or a $hile so they can o their thin'! /o a'ain it pulses the system an ma"es it re+ersible! Slide (): ?ere is the bi' picture o# $hat.s $hat 'luca'on is tellin' the cell! No$ i# you ha+e this #or a perio o# time urin' star+ation. the receptors imeri3e! The receptors phosphorylate each other. but since it oesn.re ener'i3in' this pancreas cell! (hat that oes is %T. #atty aci synthesis.nsulin is responsible #or storin' 'lucose $hen there is plenty 'lucose aroun ! *luca'on mobili3es e+erythin' because $e on.re tryin' to brea" it o$n! %lso pyru3ate kinase is phosphorylate an that bloc"s 'lycolysis! 5ou on. $hat happens4 Nothin'! 2uscle cells o not ha+e the appropriate receptor! The muscle cells ha+e all the other en3ymes. protein synthesis. microsecon s to millisecon s! *enetically it reacts hours to ays! Slide #)*: No$ i# .t ha+e a lot o# 'lucose aroun ! There is a balance o# insulin an 'luca'on in the circulatory system at all times that precisely re'ulates 'lucose le+els! /o let.sensiti+e potassium channel that lea s to epolari3ation! This opens up a +olta'e re'ulate calcium channel an allo$s calcium into the cell! Calcium bin s to an acti+ates a calcium dependent protein kinase5 &1%! The -&C phosphorylates these +esicles $hich are rich in insulin! These +esicle #use $ith the membrane an ump insulin into the circulatory system! Slide #)4: /o $hat happens $hen insulin bin s. microsecon to millisecon time #rames! -hosphatases are a little slo$er. it cannot return it to the bloo stream because it. bathe muscle cells in 'luca'on. transporter in the pancreas! /o 'lucose 'ets into pancreas cells throu'h the *01T2 transporter! *lucose 'ets in an 'ets phosphorylate an 'ets trappe an 'oes throu'h metabolism! 5ou start increasin' the %T-C%7. an in the lon' term it can lea to 'ro$th an 'ene e)pression! .s see ho$ $e 'et insulin into the system Slide #)-: /o there is GLUT.bin s to an %T. it stimulates a enylate cyclase.classes o# protein phosphatases that strip these phosphate 'roups bac" o## o# hy ro)yl 'roups an re'enerate the ori'inal protein! &inases $or" real #ast. you.re tyrosine "inases! They phosphorylate tar'et proteins on tyrosine resi ues as $ell! These tar'et proteins start a si'nal casca e that lea s to 'lyco'en synthesis. it has the receptor #or that an it $ill acti+ate the same path$ay an the muscle cell $ill mobili3e its 'lucose! %n $hat oes it o $ith the 'lucose4 .: ?ere is the insulin mo el an its si'nal trans uction casca e! .s tyrosine resi ues since they. put a little bit o# epinephrine on the cell.t $ant to be ma"in' 'lyco'en as you.t burns it. it phosphorylates some "ey tar'ets! 2ructose "isp#osp#otase starts 'luconeo'enesis! Glycogen synt#ase is phosphorylate an inhibite ! 5ou on.nsulin oes not in+ol+e secon messen'er c%2-! .s 'oin' on! 5ou ha+e 'luca'on bin in' to its receptor an acti+ates a * protein! 8nce it is acti+ate .t ha+e the receptor to tri''er e+erythin' it has no e##ect! But i# .ratio.

in a * protein. ho$ o . turn it o##4 .&11'.t has its o$n internal phosphatase acti+ity! .t also acti+ates a &16 . but you. the $hole system is shut o$n! /o it is still hormone re'ulate ! No$ these * proteins ha+e t$o classes $here one the alpha unit is e)citatory. put a *T.m 'oin' to use an inhibitory alpha subunit! . $hich turns on /= "inase.an inacti+ates itsel#! Then it $ill reassociate $ith the betaC'amma comple) to re#orm the trimer! No$ i# the hormone is still boun to the receptor.in the cell.t acti+ates *-proteins so it. the alpha helices shi#t an chan'es its interaction $ith a trimeric * protein! %n this * protein is three i##erent subunits: an alpha. an the other it is inhibitory! /o i# .an o+er the time o# 100-:00 ms.t ha+e enou'h insulin your neurons $ill ie #or a number o# reasons! Slide #)5: /o ho$ is this $or"in'4 .m 'oin' to use a stimulatory alpha subunit! . can.# . a beta..t e+entually interacts $ith an a enylate cyclase! 5ou 'et protein-protein interaction $here the alpha subunit bin s to the en3yme.t..re also chan'in' lon' term 'ro$th potential! .t $ant as much c%2. ephosphorylate 'lyco'en phosphorylase.# . on. the alpha subunit issociates #orm the betaC'amma subunit an i##uses across the inner sur#ace o# the cell membrane! . $hich turns on protein phosphatase 1! -rotein phosphatase ephosphorylates an acti+ates glycogen synt#ase an ephosphorylates an eacti+ates glycogen p#osp#atase! These path$ays are +ery lar'e an o+erlap $ith many other casca es! Slide #)): ?ere is the insulin mo el results! 5ou can rea throu'h that but the main thin' it oes is ener'y stora'e o+er ener'y mobili3ation! The 'luca'on mo el path$ay is ener'y mobili3ation o+er ener'y stora'e! Slide #+**: .# you on.T1 $hich recruits *01T4 transporters to the membrane an also acti+ates other proteins many o# $hich are #or 'ene e)pression! Slide #)!4)(: ?ere is the path$ay ra$n out! The receptor imeri3e an phosphorylate a tar'et protein @y protein . .&1'.a'ain an continue si'nalin'! 8nce this hormone lea+es or is e'ra e . $ant to tal" about protein-protein interaction an * proteins an calmo ulin! Slide #+*+: ?ere is a serotonin receptor $ith se+en transmembrane spannin' omains! . so i# .t.# you ta"e a phosphate o## 'lyco'en synthase. it hy roly3es the *T. an a 'amma! /o $hat happens is $hen a li'an bin s.s 'ot a receptor omain up here! (hen it bin s to its li'an .bac" into *7.in my cell . calle ras! Ras 'ets acti+ate an turns on a 6a# "inase! 6a# turns on myogen acti3ated protein kinase kinase $/.s its o$n en3yme! . . not only are you messin' aroun $ith the ener'etics o# cells.: 1n er this con ition.t bin s to the acti+atin' *T.t remember the name ri'ht no$A! This interacts $ith a * protein. you acti+ate an start ma"in' 'lyco'en! /o the insulin casca e uses a series o# "inases an one +ery important phosphatase! .t brea" o$n 'lyco'en anymore! .s calle a G protein coupled receptor $G&%R'! . on./o i# you ha+e a iabetic. $ant a lot o# c%2. this $ill imme iately bin *T. $hich turns on myogen acti3ated protein kinase $/.nsulin bin s to its receptor an causes auto phosphorylation $hich lea s to the acti+ation o# a phosphatase! The phosphatase is a maBor si'naler because phosphatase 'oes alon' an #in s 'lyco'en phosphorylase an 'lyco'en synthase an pulls the phosphate 'roups o##! . it stimulates the alpha subunit to pic" up a *T-! Slide #+*. inhibit it! .#rom %T-! No$ $e sai e+erythin' so #ar is re+ersible.t is a +ery common mechanism! /o here is this bun le o# se+en alpha helices! . chan'in' its con#ormation an #orms the acti+e site an #orms c%2.

s loo" at a clinical e)ample! This is 7i"rio %#olera.Slide +*-: No$ let. it causes c#olera! This causes pro#oun $atery iarrhea an +omitin' that lea s to rapi ehy ration o# the patient! %n the patient can ie $ithin 4 to D hours! The molecular mechanism in+ol+es * proteins! .