Kuliah Biokimia-Enzim FK UNDIP

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Enzymes Enzymes Compiled by :dr. Santoso



A.Introduction

B. Mechanisms of Enzymatic reactionC. Factor affecting enzyme actiity!."egulation of enzyme actiity



A.Introduction

B. Mechanisms of Enzymatic reactionC. Factor affecting enzyme actiity!."egulation of enzyme actiity



4

What Are Enzymes? What Are Enzymes? # Most enzymes are

ProteinsProteins ((tertiaryand quaternarystructures)

# Act as CatalystCatalyst toaccelerates areaction

• Not permanentlyNot permanently

changed in theprocess



5

EnzymesEnzymes# Are specific for

what they willcatalyzecatalyze

# Are ReusableReusable

# End in ase ase !"ucrase !"ucrase

!#actase !#actase

!Maltase !Maltase



A.IntroductionB. Mechanisms of Enzymatic reaction

C. Factor affecting enzyme actiity!."egulation of enzyme actiity



7

$ow do enzymes Wor%?$ow do enzymes Wor%?

Enzymes wor% bywea%enin& bondswea%enin& bonds whichwhich lowerslowers

acti'ation ener&yacti'ation ener&y



8



$ow do Enzymes Affect Reaction Rates?

Enzymes affect the ratesof reactions by lo$eringthe amount of energy ofactiation re%uired forthe reactions to begin.

&herefore processes canoccur in liing systems atlo$er temperatures orenergy leels than it$ould re%uire for these

same reactions to occur$ithout the enzymespresent.



'o$ do Enzymes Bind to Substrates

&here are t$o proposed methods by$hich enzymes bind to their substratemolecules:• (oc) and *ey Model

• Induced+Fit Model



11

Enzyme!"ubstrate ompleEnzyme!"ubstrate omple&he substancesubstance

,reactant- anenzymeenzyme acts on is

the substratesubstrate

EnzymeSubstrate oins



Acti'e "iteActi'e "ite# A restricted re&ionrestricted re&ion of an enzymeenzyme

molecule $hich bindsbinds to the substratesubstrate.

EnzymeSubstrate

Acti'e"ite



#oc% and *ey Model

enzyme

S/

S0

S0 enzyme

S/

E+,-ME "./"0RA0E1MP#E2

enzyme

"./"0RA0EM1#E.#E"

Acti'e site

1 1Products

Enzyme returns from the reaction unchan&ed

and can now react with more substrate3



4nduced 5it4nduced 5it# A chan&e in the

shapeshape of anenzyme6s acti'e

site# 4nduced4nduced by thesubstrate



4nduced 5it4nduced 5it

# A chan&echan&e in the confi&urationconfi&uration of an

enzyme6s acti'eenzyme6s acti'e sitesite ($7 and ionicbonds are in'ol'ed)3

# 4nduced4nduced by the substratesubstrate33

Enzyme

Active Sitesubstrate

induced fit



Enzyme Cooperatiity

Some enzymes haemultiple actie site. Ithas been obsered that$hen one substratemolecule binds to asingle actie site in theinactie form or tensestate of the enzyme2 aconfigurational changeoccurs in the other

actie sites ma)ing themmore receptie to othersubstrate molecules.




C. *inetic of enzyme actiity!.Factor affecting enzyme actiityE. "egulation of enzyme actiity



Enzyme *ineticsExpression for enzyme catalyzed reaction:

E + S ES E + P

k 1

k -1

k 2



Michaelis+Menten E%uation

"ate increase $ith 3S4

"ate leels off as

approach 5ma6

More S than actiesites in E

Adding S has no effect

5577 8 58 5ma6ma63S4 9 *3S4 9 *MM 3S4 3S4



# 5ma6 occurs $hen

enzyme actie sites aresaturated $ithsubstrate

# *m ,Michaelis+Menten

constant- reflectsaffinity of enzyme forits substrate

# smaller the *m2 the

greater the affinity anenzyme has for itssubstrate





h ff E ?



What Affects Enzyme Acti'ity?What Affects Enzyme Acti'ity?

# 0hree factors80hree factors8

9393 En'ironmental onditionsEn'ironmental onditions

:3:3 ofactors and oenzymesofactors and oenzymes

;3;3 Enzyme 4nhibitorsEnzyme 4nhibitors

9 E i l di i



93 En'ironmental onditions93 En'ironmental onditions

9393

EtremeEtreme

0emperature0emperature

are the mostare the most

dan&erousdan&erous

• hi&h tempshi&h temps may denature (unfold)denature (unfold)the enzyme3enzyme3

:3:3 p$p$ (most li%e < ! = p$ near neutral)(most li%e < ! = p$ near neutral)

;3;3 4onic concentration4onic concentration (salt ions)(salt ions)



&emperatureAll enzymes hae anoptimum temperature at$hich they $or) best.If you obsere theenzyme;s actiity belo$the specific

temperature it $illsteadily increase until itreaches the optimum.After the optimumtemperature is reached

the enzymes actiitydrops dramatically dueto denaturing.

>ependin& on the species theran&e of optimum acti'ity is 'erybroad3 Abo'e is a comparison ofhuman enzyme acti'ity with thatofbacteria found in hot sprin&s andoceanic 'ents3



p'

All enzymes hae an

optimum p' at $hichthey $or) best. Ifthe p' falls belo$ orrises aboe theoptimum alue2enzymatic actiitydecreases

as a result ofdenaturing.

4n the human body6s di&esti'e tractthere are 'ariations in p$ from areato area3 0he stomach6s @uices6 p$

is around : (acidic) the enzyme pepsinfound in the &astric @uices hasoptimum acti'ity at a p$ of :3 0he smallintestine6s @uice6s p$ is around = (basic)30he enzyme trypsin found in the smallintestine6s @uices has optimum acti'ity at a p$of =3



Substrate Concentration

0he concentration of substrate also has an affect on the rateof enzyme acti'ity3 4f the concentration of substrate isincreased while the concentration of enzyme is constant thele'el of enzyme acti'ity will increase until a point of saturationis reached3 At this point there are no enzymes a'ailable toreact with ecess substrate and the rate of the reactionstabilizes3 +o matter if you continue to add substrate thereaction rate will not increase

4ncreasin& "ubstrate oncentration

Rate of Reaction Point of "aturation all acti'esites are filled with substrate3

: f d : f t d



:3 ofactors and oenzymes:3 ofactors and oenzymes

#4nor&anic substances4nor&anic substances

(zinc iron)(zinc iron)

and

'itamins'itamins (respecti'ely) are sometimes needfor proper enzymatic acti'ityenzymatic acti'ity3

# Eample8Eample84ron4ron must be present in the quaternaryquaternary

structurestructure !! hemo&lobinhemo&lobin in order for it to pic%pic%

up oy&en3up oy&en3



Coenzymes are bound at the actie site in order tointeract $ith the substrate and play an essential role inthe catalysed reaction.&hey act as carriers of a ariety of chemical groups.



Most water!soluble 'itamins are components of coenzymes

Bitamin oenzyme >eficiency

&hiamine ,B/-&hiamine

pyrophosphate

Beriberi ,$eight

loss2other problems

"iboflain ,B0-

FA!

Mouth lesions2 dermatitis<icotinic acid

,niacine-<A! 1ellagra ,dermatitis2

depression-

1antohtinic acid Coenzyme A 'ypertension

Biotin Biotin "ash2 muscle pain

; E 4 hibit



;3 Enzyme 4nhibitors

Specific for an enzyme

Can be reersible or non+reersible

Competitie inhibitors<on+competitie inhibitors

titi i hibit titi i hibit



31

ompetiti'e inhibitorsompetiti'e inhibitors

chemicals that resembleresemble an enzyme6senzyme6snormal substratenormal substrate and competecompete withit for the acti'e siteacti'e site3

Enzymeompetiti'e inhibitor

Substrate

+ titi 4 hibit+ titi 4 hibit



32

+oncompetiti'e 4nhibitors+oncompetiti'e 4nhibitors

Inhibitors that do not enter thedo not enter the actie siteactie site2 but

bind tobind to another partanother part of the enzymeenzyme causing theenzymeenzyme to change its shapechange its shape2 $hich in turnalters the actie sitealters the actie site.

Enzymeacti'e site

altered

+oncompetiti'e4nhibitor

Substrate

C i i N i i i hibi



Competitive vs. Non-competitive inhibitors







Enzyme acti'ity is re&ulated by four

different mechanismsC(9) Allosteric control

(:) o'alent modification

(;) Proteolytic acti'ation(D) "timulation and inhibition by control proteins

C chan&es in enzyme le'els due to re&ulation of

protein synthesis or de&radation are additionallon&!term ways to re&ulate enzyme acti'ity

Allosteric re&ulation of enzyme acti'ity



Allosteric re&ulation of enzyme acti'ity

Allosteric re&ulation the acti'ation orinhibition of an enzyme6s acti'ity due to bindin&of an effector molecule at a re&ulatory sitethat is distinct from the acti'e site of theenzyme

Allosteric re&ulators &enerally act by increasin&or decreasin& the enzyme6s affinity for thesubstrate

ll i l i



Allosteric regulation

Many allosterically controlled enzymse sho$%uaternary structure

o'alent modification re&ulates the catalytic



o'alent modification re&ulates the catalyticacti'ity of some enzymes

Can either activate it or inhibit it by altering the

conformation of the enzyme or by serving as a

functional group in the active site.

Enzyme

Modifyinggroup

EnzymeModifyinggroup

Inactive Enzyme Active Enzyme

.



Phosphorylation ! an eample of re&ulation by

re'ersible co'alent modification of the enzyme

4nsertin& a ne&ati'ely char&ed phosphate &roup

into the appropriate location in an enzyme caninduce a conformational chan&e in the enzyme thateither increases or decreases its acti'ity3

O

!!

O "O#

O#

OA$" A%"



0op F reasons why phosphorylation is used

to re&ulate enzyme acti'ity893 Phosphorylation is rapidly re'ersible ma%in& it possible to quic%lyswitch between acti'e and inacti'e forms of an enzyme3

:3 Phosphorylation is relati'ely inepensi'e since it does not requirethe synthesis of new protein molecules3

;3 Results in lar&e GH rn for the phosphorylation reaction3Phosphorylation can shift the conformational equilibrium of aprotein by a factor of I9JD3

D3 PhosphorylationKdephosphorylation is rapid and its timin& can bead@usted to meet the physiolo&ical needs of the cell3

F3 Phosphorylation effects can be rapidly amplified 'ia a %inasecascade3



"ummary8 o'alent modification

93 o'alent modification allows an enzyme to be rapidlyacti'ated or inacti'ated

:3 With co'alent modification re&ulation of a enzymeacti'ity is achie'ed at low ener&y costs to the cell (i3e3re&ulation does not require synthesis of a new enzyme orinhibitory protein)3

;3 Phosphorylation is a &ood eample of how enzymes areacti'ated and inacti'ated by co'alent post!translationalmodifications



Proteolytic acti'ation




"uch as those in'ol'ed in protein di&estion blood clottin& and boneand tissue remodelin& must be %ept in a completely inacti'e stateuntil they are needed3 0hese enzymes are synthesized as inacti'e

precursors (%nown as zymo&ens or proenzymes) and acti'ated whenneeded by proteolytic clea'a&e of a specific peptide bond in thezymo&en3

.

Enzyme &inactive'

Enzyme &active'

"ropeptide

Proteolytic Enzyme

"

r o

p

e p

t i d

e


Re&ulation of di&esti'e enzymes



Re&ulation of di&esti'e enzymes

Val (Asp) Lys Ile Val

Val (Asp) Lys Ile Val

Trypsinogen

+

Enteropeptidase

Trypsin4

4

Proteolytic activation

of digestive enzymes



>i&estion of proteins requires simultaneousacti'ation of se'eral di&esti'e enzymes3

0his is achie'ed by synthesizin& the di&esti'eenzymes as inacti'e zymo&ens that are acti'atedby specific proteolysis by trypsin3

0rypsin is acti'ated by enteropeptidasecatalyzed proteolysis of a unique lysine!isoleucine peptide bond (this is the Lmasterswitch that turns on the acti'ation of the

di&esti'e enzymes)3

Pepsino&en is con'erted to pepsin by



Pepsino&en is con'erted to pepsin byautocatalytic proteolysis at p$ :

•

Pepsino&en has a lowamount of acti'ity atp$ : allowin& it toclea'e the peptidebind between aminoacids D; and DD to&enerate pepsin thatis much more acti'ethan pepsino&en3

pepsinogen

&inactive'

pepsin &active'

Secretion intostomach &p ('

autocatalyticcleavage of pepsinogen after amino acid ))



,ymo&en

Pepsino&enhymotrypsino&en0rypsino&enProcarboypeptidaseProelastaseProthrombin

5ibrino&en5actor B445actor 2ProinsulinProcolla&enProcolla&enase

Acti'e Enzyme

Pepsinhymotrypsin0rypsinarboypeptidaseElastase0hrombin

5ibrin5actor B44a5actor 2a4nsulinolla&enolla&enase

5unction

protein di&estionprotein di&estionprotein di&estionprotein di&estionprotein di&estionblood clot formation

blood clot formationblood clot formationblood clot formationplasma &lucosehomeostasiscomponent of s%in and

bone remodelin&processes durin&metamorphosis etc3



>i&esti'e enzymes blood clottin& enzymes and enzymes

in'ol'ed in bone and tissue remodelin& catalyze reactionsthat would be disastrous if they occurred atinappropriate times or locations3

5or eample if proteolytic di&estion of proteins

occurred in the pancreas they would start di&estin&the pancreas itself3 "imilarly if blood clottin& factorsare acti'ated when they aren6t needed they willinitiate blood clottin& throu&hout the body3

"o they are synthesized as inacti'e zymo&ens and arestored in this inacti'e state until they are needed3

/lood clot formation an eample of zymo&en acti'ations



/lood clot formation ! an eample of zymo&en acti'ations

%amaged Surface

*II

+ininogen+alli,rein

*IIa

*I *Ia

I* I*a

-IIIa

* *a

-a

"rothrombin $hrombin

*IIIa

ibrinogen ibrin

Cross#lin,ed fibrin clot

-II-IIa

*

$issue

factor

$rauma

Intrinsic "ath/ay

E0trinsic "ath/ay



/lood clottin& is an ecellent eample of a proteolyticcascade desi&ned to amplify an eternal si&nal (e3&3

trauma) and e'o%e a rapid response (blood clot

formation)3

0hrombin itself is inhibited by antithrombin (a serpin)3

0his pro'ides the body with a mechanism to pre'ent

random blood clot formation beyond the site of in@ury3

Proteolytic clea'a&e differs from phosphorylation



93 4t can occur outside of cells since A0P is not

needed to con'ert the zymo&en into the acti'e

form of the enzyme3

:3 4t is not a re'ersible reaction3 4nacti'ation of theacti'e enzyme must occur by either de&radation of

the enzyme or by inhibition (e3&3 due to the bindin&

of an inhibitory protein to the acti'e enzyme)3

Proteolytic clea'a&e differs from phosphorylation

"timulation and inhibition by control proteins



"timulation and inhibition by control proteins

"ome enzymes ha'e re&ulatory proteins that bind to themand re&ulate their acti'ity3 cAMP!dependent protein %inaseis one eamples of this type of re&ulation3

Enzyme&active'

Enzyme

&inactive'Inhibitory "rotein

Inhibitory "rotein

"erpins ! An eample of inhibition by control proteins



0rypsin (oran&e) bound to bo'ine pancreatic trypsin inhibitor('iolet)3 $is FN Asp 9J: Hly 9O; and "er 9OF in theacti'e site of trypsin are shown in &reen red cyan and bluerespecti'ely3 #ys 9F in /P04 forms a salt brid&e with Asp 9=Oin trypsin in the trypsin8/P04 comple3 /indin& of bo'inepancreatic trypsin inhibitor is essentially irre'ersible.

"erpins ! An eample of inhibition by control proteins

1nce trypsin is acti'ated weneed a mechanism to turn it

off when it is no lon&erneeded3 Pancreatic trypsininhibitor is used to shut offtrypsin acti'ity – pancreatic trypsin inhibitor

binds 'ery ti&htly to trypsin –

pancreatic trypsin inhibitoris a member of a class ofproteins %nown as serineprotease inhibitors (serpins)3

– "erpins are polypeptides

that inhibit serine proteasesby bindin& to the acti'esites of these enzymes3

El h b d b 9



Elastase is inhibited by α9!antitrypsin

α 9!antitrypsin inhibits elastase aserine protease that is responsiblefor remodelin& colla&en3

4ndi'iduals in which Hlu ;D: in α9!

antitrypsin is replaced by a lysinesecrete only 9F of the normalle'els for α 9!antitrypsin resultin&in uncontrolled elastase acti'ity andthe brea%down of the al'eolar wallsin the lun&3



+ote that althou&h serpins are ti&ht bindin&

inhibitors or serine proteases they do not form

co'alent bonds with the serine proteases3

4n other words bindin& of serpins to serineproteases does not in'ol'e the formation of co'alent

bonds between the serpins and the serine proteases3

"ummary of re&ulatory mechanisms



"ummary of re&ulatory mechanisms

93 Allosteric re&ulation

A0P acti'ationK0P inhibition of A0ase si&moidal %inetics

cAMP acti'ation of cAMP!dependent protein %inase

:3 Re'ersible co'alent modification

Phosphorylation

"erK0hr protein %inases 0yr %inases %inase cascades

;3 Proteolytic acti'ation

>i&esti'e enzyme blood clottin& factors

D3 Protein acti'ators and inhibitors"erpins

Re&ulatin& the rates of enzyme!dri'en



& & f ymreactions

Cells use inhibitors and actiators to turn off andon enzymes

Many enzymes are controlled by an allosteric siteremote from the actie site

Feedbac) inhibition



Enzyme / Enzyme 0 Enzyme =Inter+

mediateInter+

mediate>1roduct

Start ofpath$ay

1resence of product inhibits enzyme /

Feedbac) inhibition

Many enzymes are actually regulated by the

end products of the reaction they catalyze

&his preents too much product from being made

An e6ample of Feedbac) inhibition



p

&his e6ample demonstrates ho$ anend product can inhibit the first

step in its production. Isoleucinebinds to the allosteric site ofthreonine deaminase and preentsthreonine from binding to the actiesite because the shape of the actie

site is altered. ?hen the leel ofisoleucine drops in the cell;scytoplasm2 the isoleucine is remoedfrom the allosteric site on theenzyme2 the actie site resumes the

actiated shape and the path$ay is@cut bac) on and isoleucine beginsto be produced.



Klasifikasi Enzim

Sistem Penamaan Enzim

Spesifitas Enzimacam!macam "entuk Enzim

Klasifikasi Enzim



Klasifikasi Enzim

# kelas

$ksidoreductase

%ransferase

&idrolase

'somerase(igase





)eaksi dan enzim yang mengkatalisisnya membentuk enam

kelas *+ hingga ,- subkelas

/ bagian : 0ama pertama substratnya

0ama kedua tipe reaksi yang dikatalisis.

'nformasi tambahan1 dituliskan dalam tanda kurung di bagian

akhir2

misalnya enzim yang mengkatalisis reaksi (!malat 3 045

3

6 piruvat3 C$/ 3 045& 3 &3 diberi nama ,.,.,.-7 (!malat:0453

oksidoreduktase *dekarboksilasi

Setiap enzim mempunyai nomor kode *EC yang menandai tipe

reaksi berkenaan dengan

kelas *digit pertama

subkelas *digit kedua

subsubkelas *digit ketiga

Spesifitas Enzim



Spesifitas Enzim

Enzim biasanya sangat spesifik dalam aksinya.

"eberapa kespesifikan yang dimiliki oleh enzim antara lain :

Kespesifikan geometrik

Kespesifikan reaksi

Kespesifikan optik.

Kespesifikan organella

Macam-macam Bentuk Enzim



Macam macam Bentuk Enzim

Proenzim

"entuk enzim yang inkatif

'sozim

"entuk enzim berbeda yang mengkatalisis reaksi kimia yang

sama. 'sozim ini berasal dari duplikasi gen

4losterik enzim"entuk enzim yang diatur dengan mekanisme alosterisme.

4da / sisi :

Sisi aktif

Sisi regulatorik



nzim plasma

fungsional

nzim plasma non

fungsional

Konsentrasi

dalam

plasma

(ebih tinggi dalam

plasma dibandingkan

dengan di8aringan

Secara normal1 konsentrasi

di dalam plasma sangat

rendah dibandingkan dengan

di dalam 8aringan

9ungsi elas %idak 8elas

Substrat "erada dalam darah %idak ada dalam darah%empat

sintesis

&epar 5iberbagai macam organ

seperti hepar1 8antung1 otak

dan otot rangka

Contoh 9actor pembekuan

(ipoprotein lipase

Pseudocholine

esterase

4(%1 4S%1 CK1 (5&1 alkaline

phospatase1 amilase



&'A<* D

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