13 14 Biochemistry Elliot Enzymes Regulation and Signal Transduction 01-10-14

8/12/2019 13 14 Biochemistry Elliot Enzymes Regulation and Signal Transduction 01-10-14

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BiochemistryElliot

January 10, 201410:00pm-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 yiels 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! Thats a si'niicant

alteration in a protein surace! This mutation occurs on $hats calle the plekstrinbinin'

omain! lec"strin is an aaptor protein that allo$s t$o proteins to bin to'ether! The mutation

o plec"strin allo$s proteins to bin to it constantly! . its boun to plec"strin constantly, its

'oin' to bin to the "inase an allo$ it to interact $ith GLUT4 transporters, an recruit them to

the membrane constantly! /o youre 'oin' to ha+e *T4 transporters in muscle an aipose

in the membrane constantly! Theyre not suppose to be there, there only suppose to be there

in times o plenty! /o muscle an aipose ta"e up all the 'lucose in the bloo an lea+e little or

the brain, $hite bloo cells, an re bloo cells!

/o *T4 is there all the time! uscle is constantly ta"in' 'lucose out! /o you 'o to be an

your bloo 'lucose $hile youre astin' comes o$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

*T4 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 prooun hypo'lycemia!

/o ho$ o you compensate or that 5ou can eat comple) corn starch beore 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 757!: Ne)t thin' $ere tal"in' about is allosteric control! This is ho$ you control a

comple) en3yme! (e are 'oin' to loo" at this control o aspartate transcar"amoylase$hich

produces cytidine tripospate $%T&'! .t is the rate limitin' step in this reaction!

Slide 77: This is the crystalline structure o the en3yme reuce to alpha helices an beta

sheets! There are si) catalytic subunits, there are si) re'ulatory subunits! /o $hat happens

here is $ere only at physiolo'ical substrate concentration here! /o ichaelis-enten '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 $ino$ o substrate

concentration in a li+in' cell! /o the concentration o aspartate is 'oin' to be in a narro$ ran'e!

Thereore . ha+e to i'ure out a $ay to re'ulate the en3yme so that it has a hi'her ainity or thesubstrate because . cant 'et the cell to increase aspartate on its o$n! /o . ha+e to mess $ith

the en3yme to impro+e its ainity or $hat substrate is there! %n thats $hat this is about! 5ou

'o rom this ichaelis cur+e to a si'moial cur+e! 5ou start to bin substrate an then you can

bin a $hole lot o substrate! 5ou start to release prouct, you release a $hole lot o prouct!

Slide 7(: /o this is $hat the structure loo"s li"e! %n ho$ it $or"s is a monument to structural

biolo'y! ner tense non-reacti+e catalytic conitions, theses si) catalytic subunits collapse


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o$n on themsel+es! /o its nice an pac"e! The en prouct, cytiine triphosphate, the

prouct o this lon' path$ay comes bac" an bins to an allosteric site! %n allosteric site is a

speciic binin' site o CT that occurs on the re'ulatory subunit, it oes not occur on the

catalytic subunit! But CT bins to this allosteric binin' site an chan'es the conormation o

the re'ulatory subunit $hich is connecte to the catalytic subunit an causes the entire comple)

to collapse on itsel!

No$ i CT le+els ecrease, because youre usin' them to ma"e 6N% or 7N%, an you nee to

ma"e more o this stu! These sites are not 'oin' to be occupie at $hich point they chan'e

conormation an the comple) e)pans or opens! (hats 'oo about that is the acti+e sites are

on the internal surace! (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! . the acti+e site is bloc"e, you cant 'et

substrate into it, not substrate, no reaction, an reaction rates rop ramatically!

/o i . ha+e plenty o CT then the substrates are bloc"e an .m not ma"in' any more prouct

because . ha+e plenty!

There are also allosteric acti+ators! %T! . there is a lot o %T it tells you t$o thin's! 8ne,

theres a lot o ener'y, lets use it to buil somethin'! T$o, theres a lot o purine nucleoties,an $e $ant to ma"e about an e9ual molar amount o pyrimiine nucleoties! /o *T an %T

are purines an you $ant about an e9ual amount o pyrimiines, , T, an C! That $ay, your

7N% an 6N% polymerases $or" more eiciently! /o i . ha+e a lot o %T, . $ant to ma"e a lot

o pyrimiines to balance it all o! /o theres another site on the re'ulatory omain that bins

%T! (hen the %T bins, it orces this comple) open! /o its an allosteric acti+ator!

Slide #7): /o i you put in a little %T, you acti+ate the system, i you put a little CT it

eacti+ates the system! %n you $ant to loo" at this i'ure here, you can see i . put in some

%T . increase the reaction rate about a 1000 ol! . . put in some CT . can rop the reaction

rate to about ;! %'ain these thin's are not all on or all o! .ts in ynamic e9uilibrium so there

is constant ine tunin' 'oin' on!

Slide #(*(+: 8" no$ .ll tal" about co+alent moiication by proteolysis! 7i'esti+e proteases,

amylases, an lipases are mae as pre-en3ymes or 3ymo'ens: inacti+e! Entero"inases in the

'ut linin' sees trypsinogensan clips it an the $hole system reor'ani3es into the acti+e

ormation! %t this point, the acti+e trypsinsees other tar'et proteins li"e chymotrypsino'en,

elastino'en! % lot o these proen3ymes 'et clea+e at speciic bons that allo$ them all to

reor'ani3e an orm the acti+e site! /o here is cymotrypsinogenan it is clea+e here

bet$een resiues 1< an 1=, an 14 an 14> to orm cymotrypsin! This is +ery common!

Slide: (,: &epsinogenis ma"e in the stomach linin', 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 itsacti+e site an cuts itsel! Then it reor'ani3es to that it cut other aci enature ietary proteins!

Slide (-: ?ere is the clottin' cascae! 5ou o not nee to "no$ the clottin' cascae ri'ht no$!

But $hat . $ante to sho$ you is that e+ery en3yme here is a serine protease, but as a

3ymo'en or inacti+e!

Slide (4(5: %nother $ay o re'ulatin' en3ymes is co+alent moiication! ets 'o bac" to

"inases an $hat they o an ho$ they $or"! Glucagonis mae by the pancreas $hen


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'lucose le+els are lo$! .ts a small polypeptie! This polypeptie has a really hi'h ainity or a

receptor!

Slide (!: /o 'luca'on @or epinephrineA bins to its speciic receptor! The receptor chan'es

conormation $hich is transmitte to a si'nal transuction protein! .n this case the si'nal

transuction protein is a G protein! * stans or GT&so this is a *T binin' protein! .n the

inacti+e state the * protein is boun to *7! 8nce the li'an bins to the receptor, the receptorchan'es its conormation, $hich inuces a chan'e in conormation o the * protein or a much

hi'her ainity or *T! /o *T comes in an islo'es the *7! This is the acti+ate protein!

This acti+ate * protein oc"s $ith a tar'et protein! .n this case the tar'et protein is adenylate

cyclase! %enylate cyclase, once it oc"s $ith a * protein is turne on an turns %T into

c./&! c% is some$hat unstable an e'raes, but its tar'et proteins reco'ni3e it an start

a $hole bunch o si'nalin' cascaes +ery rapily! /o . ha+e a li'an that bins to a receptor

that acti+ates a transuction protein that bins to an en3yme that creates a prouct that turns on

a si'nalin' cascae!

7espite c% bein' unstable, it has a protein that brea"s it o$n! &ospodiesterase! .t turns

c% to %! 5ou nee this because $hen you si'nal a system to o somethin', you $ant topulse it! ulsin' allo$s you to o ine tunin' increments o $hate+er metabolism you $ant to

aust to accommoate the en+ironment! . you int 'et ri o this c% you $oul be

recruitin' a $hole more o these cyclic systems, $hich may inuce inappropriate metabolism or

'ene e)pression!

%a00eineinhibits phosphoiesterase! . . inhibit phosphoiesterase, c% stays aroun a lot

lon'er! /o this protein kinase . $&1.''ets acti+ate or a lon'er perio o time! .t

phosphorylates its tar'ets or a lon'er perio o time! 8ne o the tar'ets is a soium transporter,

so more soium lea+es the cell! . more soium lea+es the cell more chlorie lea+es the cell

$hich causes more $ater to lea+e the cell! (hy o you thin" e)a) has so much caeine in it

. you ha+e a lot o caeine in your system, you pump out more soium an $ater into your

intestinal tract!

Slide #(7: &% is actually a tetramer $ith t$o re'ulatory subunits! c% bins to these an

they lose ainity or the catalytic subunits $hich acti+ates &%! (hen c% is hyroly3e to

%, the re'ulatory subunits re'ain their ainity 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 or *T

an phosphorylates other proteins! ?ere a "inase is acti+ate an attaches a phosphate to an

alcohol 'roup! There are t$o classes! There are serinetreonine kinasesthat stic" a

phosphate on a serine or threonine an tyrosine "inases! The tyrosine kinaseamily typically

ten to be membrane receptor proteins! The serinethreonine "inases ten to by ree-roamin',

cytosolic "inases!

?ere . ha+e a protein that becomes inacti+e $hen . phosphorylate it, but it can easily 'o the

other $ay! . can acti+ate it $hen . put a phosphate on! .n act, they e)ist! 5oull see in the case

or insulin . phosphorylate a protein to acti+ate 'lyco'en brea"o$n, an phosphorylate another

protein to inacti+ate 'lycolysis! /o i . use one si'nal, phosphorylate these proteins, . acti+ate

one system an inacti+ate the other simultaneously! /o this is nicely re+ersible! There are t$o


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classes o protein phosphatases that strip these phosphate 'roups bac" o o hyro)yl 'roups

an re'enerate the ori'inal protein!

&inases $or" real ast, microsecon to millisecon time rames! hosphatases are a little

slo$er, li"e tenol slo$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 $hats 'oin' on! 5ou ha+e 'luca'on binin' to its receptor

an acti+ates a * protein! 8nce it is acti+ate, it stimulates aenylate cyclase, $hich ma"es

c%! c% acti+ates &% an this oes a number o thin's, it phosphorylates some "ey

tar'ets! 2ructose "ispospotasestarts 'luconeo'enesis! Glycogen syntaseis

phosphorylate an inhibite! 5ou ont $ant to be ma"in' 'lyco'en as youre tryin' to brea" it

o$n! %lso pyru3ate kinaseis phosphorylate an that bloc"s 'lycolysis! 5ou ont $ant to

burn 'lucose $hen it is lo$! Thats $hat 'luca'on is tellin' the cell!

No$ i you ha+e this or a perio o time urin' star+ation, &% $ill acti+ate proteins or 'ene

e)pression so you can 'et lon' term recruitment o en3ymes neee to support

'luconeo'enesis! /o metabolically this system reacts 9uic"ly, microsecons to millisecons!

*enetically it reacts hours to ays!

Slide #)*: No$ i . bathe muscle cells in 'luca'on, $hat happens Nothin'! uscle cells o not

ha+e the appropriate receptor! The muscle cells ha+e all the other en3ymes, but since it oesnt

ha+e the receptor to tri''er e+erythin' it has no eect! But i . put a little bit o epinephrine on

the cell, 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 'lucose .t burns it, it cannot return it to the

bloostream because its missin' an en3yme! Glucose!pospatestays as 'lucose-=-

phosphate so it is trappe in the cell! uscle cells cannot ta"e that phosphate 'roup o li"e li+er

cells can!

Slide #)+),: ?ere is the insulin moel an its si'nal transuction cascae! .nsulin oes not

in+ol+e secon messen'er c%! .nsulin also acts throu'h its o$n tyrosine "inase receptor antar'ets a lot o ierent proteins! .nsulin is responsible or storin' 'lucose $hen there is plenty

'lucose aroun! *luca'on mobili3es e+erythin' because $e ont 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 lets see ho$ $e 'et insulin into the system

Slide #)-: /o there is GLUT,transporterin the pancreas! /o 'lucose 'ets into pancreas cells

throu'h the *T2 transporter! *lucose 'ets in an 'ets phosphorylate an 'ets trappe an

'oes throu'h metabolism! 5ou start increasin' the %T%7 ratio, youre ener'i3in' this

pancreas cell! (hat that oes is %T bins to an %T sensiti+e potassium channel that leas to

epolari3ation! This opens up a +olta'e re'ulate calcium channel an allo$s calcium into the

cell! Calcium bins to an acti+ates a calcium dependent protein kinase &1%! The &C

phosphorylates these +esicles $hich are rich in insulin! These +esicle use $ith the membranean ump insulin into the circulatory system!

Slide #)4: /o $hat happens $hen insulin bins, the receptors imeri3e! The receptors

phosphorylate each others tyrosine resiues since theyre tyrosine "inases! They phosphorylate

tar'et proteins on tyrosine resiues as $ell! These tar'et proteins start a si'nal cascae that

leas to 'lyco'en synthesis, atty aci synthesis, protein synthesis, an in the lon' term it can

lea to 'ro$th an 'ene e)pression!


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/o i you ha+e a iabetic, not only are you messin' aroun $ith the ener'etics o cells, but

youre also chan'in' lon' term 'ro$th potential! . you ont ha+e enou'h insulin your neurons

$ill ie or a number o reasons!

Slide #)5: /o ho$ is this $or"in' .nsulin bins to its receptor an causes auto phosphorylation

$hich leas to the acti+ation o a phosphatase! The phosphatase is a maor si'naler because

phosphatase 'oes alon' an ins 'lyco'en phosphorylase an 'lyco'en synthase an pullsthe phosphate 'roups o! . . ephosphorylate 'lyco'en phosphorylase, . inhibit it! . ont brea"

o$n 'lyco'en anymore! . you ta"e a phosphate o 'lyco'en synthase, you acti+ate an start

ma"in' 'lyco'en! /o the insulin cascae uses a series o "inases an one +ery important

phosphatase! .t also acti+ates a &16.T1$hich recruits *T4 transporters to the membrane

an also acti+ates other proteins many o $hich are or 'ene e)pression!

Slide #)!)(: ?ere is the path$ay ra$n out! The receptor imeri3e an phosphorylate a tar'et

protein @y protein . cant remember the name ri'ht no$A! This interacts $ith a * protein, calle

ras! Ras'ets acti+ate an turns on a 6a "inase! 6a turns on myogen acti3ated protein

kinase kinase $/.&11', $hich turns on myogen acti3ated protein kinase $/.&1', $hich

turns on /= "inase, $hich turns on protein phosphatase 1! rotein phosphatase

ephosphorylates an acti+ates glycogen syntasean ephosphorylates an eacti+ates

glycogen pospatase! These path$ays are +ery lar'e an o+erlap $ith many other

cascaes!

Slide #)): ?ere is the insulin moel 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 moel path$ay is ener'y

mobili3ation o+er ener'y stora'e!

Slide #+**: . $ant to tal" about protein-protein interaction an * proteins an calmoulin!

Slide #+*+: ?ere is a serotonin receptor $ith se+en transmembrane spannin' omains! .t

acti+ates *-proteins so its calle a G protein coupled receptor $G&%R'! .t is a +ery common

mechanism! /o here is this bunle o se+en alpha helices! .ts 'ot a receptor omain up here!(hen it bins to its li'an, the alpha helices shit an chan'es its interaction $ith a trimeric *

protein! %n this * protein is three ierent subunits: an alpha, a beta, an a 'amma! /o $hat

happens is $hen a li'an bins, it stimulates the alpha subunit to pic" up a *T!

Slide #+*,: ner this conition, the alpha subunit issociates orm the beta'amma subunit

an iuses across the inner surace o the cell membrane! .t e+entually interacts $ith an

aenylate cyclase! 5ou 'et protein-protein interaction $here the alpha subunit bins to the

en3yme, chan'in' its conormation an orms the acti+e site an orms c% rom %T! No$

$e sai e+erythin' so ar is re+ersible, so i . put a *T in a * protein, ho$ o . turn it o .t has

its o$n internal phosphatase acti+ity! .ts its o$n en3yme! .t bins to the acti+atin' *T an

o+er the time o 100-00 ms, it hyroly3es the *T bac" into *7 an inacti+ates itsel! Then it

$ill reassociate $ith the beta'amma comple) to reorm the trimer!

No$ i the hormone is still boun to the receptor, this $ill immeiately bin *T a'ain an

continue si'nalin'! 8nce this hormone lea+es or is e'rae, 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, an the other it is

inhibitory! /o i . $ant a lot o c% in my cell .m 'oin' to use a stimulatory alpha subunit! . .

ont $ant as much c% in the cell, .m 'oin' to use an inhibitory alpha subunit!


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Slide +*-: No$ lets loo" at a clinical e)ample! This is i"rio %olera, it causes colera! This

causes prooun $atery iarrhea an +omitin' that leas to rapi ehyration o the patient!

%n the patient can ie $ithin 4 to D hours! The molecular mechanism in+ol+es * proteins!

Documents

13 14 Biochemistry Elliot Enzymes Regulation and Signal Transduction 01-10-14