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Enzymes Enzymes Compiled by :dr. Santoso
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A.Introduction
B. Mechanisms of Enzymatic reactionC. Factor affecting enzyme actiity!."egulation of enzyme actiity
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A.Introduction
B. Mechanisms of Enzymatic reactionC. Factor affecting enzyme actiity!."egulation of enzyme actiity
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What Are Enzymes? What Are Enzymes? # Most enzymes are
ProteinsProteins ((tertiaryand quaternarystructures)
# Act as CatalystCatalyst toaccelerates areaction
• Not permanentlyNot permanently
changed in theprocess
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EnzymesEnzymes# Are specific for
what they willcatalyzecatalyze
# Are ReusableReusable
# End in ase ase !"ucrase !"ucrase
!#actase !#actase
!Maltase !Maltase
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A.IntroductionB. Mechanisms of Enzymatic reaction
C. Factor affecting enzyme actiity!."egulation of enzyme actiity
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$ow do enzymes Wor%?$ow do enzymes Wor%?
Enzymes wor% bywea%enin& bondswea%enin& bonds whichwhich lowerslowers
acti'ation ener&yacti'ation ener&y
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$ow do Enzymes Affect Reaction Rates?
Enzymes affect the ratesof reactions by lo$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$ithout the enzymespresent.
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'o$ 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
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Enzyme!"ubstrate ompleEnzyme!"ubstrate omple&he substancesubstance
,reactant- anenzymeenzyme acts on is
the substratesubstrate
EnzymeSubstrate oins
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Acti'e "iteActi'e "ite# A restricted re&ionrestricted re&ion of an enzymeenzyme
molecule $hich bindsbinds to the substratesubstrate.
EnzymeSubstrate
Acti'e"ite
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#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
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4nduced 5it4nduced 5it# A chan&e in the
shapeshape of anenzyme6s acti'e
site# 4nduced4nduced by thesubstrate
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4nduced 5it4nduced 5it
# A chan&echan&e in the confi&urationconfi&uration of an
enzyme6s acti'eenzyme6s acti'e sitesite ($7 and ionicbonds are in'ol'ed)3
# 4nduced4nduced by the substratesubstrate33
Enzyme
Active Sitesubstrate
induced fit
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Enzyme Cooperatiity
Some enzymes haemultiple actie site. Ithas been obsered that$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.
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A.IntroductionB. Mechanisms of Enzymatic reaction
C. *inetic of enzyme actiity!.Factor affecting enzyme actiityE. "egulation of enzyme actiity
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Enzyme *ineticsExpression for enzyme catalyzed reaction:
E + S ES E + P
k 1
k -1
k 2
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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
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# 5ma6 occurs $hen
enzyme actie sites aresaturated $ithsubstrate
# *m ,Michaelis+Menten
constant- reflectsaffinity of enzyme forits substrate
# smaller the *m2 the
greater the affinity anenzyme has for itssubstrate
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A.IntroductionB. Mechanisms of Enzymatic reaction
C. Factor affecting enzyme actiity!."egulation of enzyme actiity
h ff E ?
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What Affects Enzyme Acti'ity?What Affects Enzyme Acti'ity?
# 0hree factors80hree factors8
9393 En'ironmental onditionsEn'ironmental onditions
:3:3 ofactors and oenzymesofactors and oenzymes
;3;3 Enzyme 4nhibitorsEnzyme 4nhibitors
9 E i l di i
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93 En'ironmental onditions93 En'ironmental onditions
9393
EtremeEtreme
0emperature0emperature
are the mostare the most
dan&erousdan&erous
• hi&h tempshi&h temps may denature (unfold)denature (unfold)the enzyme3enzyme3
:3:3 p$p$ (most li%e < ! = p$ near neutral)(most li%e < ! = p$ near neutral)
;3;3 4onic concentration4onic concentration (salt ions)(salt ions)
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&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
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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
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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
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: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
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Coenzymes are bound at the actie site in order tointeract $ith the substrate and play an essential role inthe catalysed reaction.&hey act as carriers of a ariety of chemical groups.
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Most water!soluble 'itamins are components of coenzymes
Bitamin oenzyme >eficiency
&hiamine ,B/-&hiamine
pyrophosphate
Beriberi ,$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
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;3 Enzyme 4nhibitors
Specific for an enzyme
Can be reersible or non+reersible
Competitie inhibitors<on+competitie inhibitors
titi i hibit titi i hibit
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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
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+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 $hich in turnalters the actie sitealters the actie site.
Enzymeacti'e site
altered
+oncompetiti'e4nhibitor
Substrate
C i i N i i i hibi
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Competitive vs. Non-competitive inhibitors
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A.IntroductionB. Mechanisms of Enzymatic reaction
C. Factor affecting enzyme actiity!."egulation of enzyme actiity
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Enzyme acti'ity is re&ulated by four
different mechanismsC(9) Allosteric control
(:) o'alent modification
(;) Proteolytic acti'ation(D) "timulation and inhibition by control proteins
C chan&es in enzyme le'els due to re&ulation of
protein synthesis or de&radation are additionallon&!term ways to re&ulate enzyme acti'ity
Allosteric re&ulation of enzyme acti'ity
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Allosteric re&ulation of enzyme acti'ity
Allosteric re&ulation the acti'ation orinhibition of an enzyme6s acti'ity due to bindin&of an effector molecule at a re&ulatory sitethat is distinct from the acti'e site of theenzyme
Allosteric re&ulators &enerally act by increasin&or decreasin& the enzyme6s affinity for thesubstrate
ll i l i
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Allosteric regulation
Many allosterically controlled enzymse sho$%uaternary structure
o'alent modification re&ulates the catalytic
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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
.
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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%"
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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
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"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
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Proteolytic acti'ation
Proteolytic acti'ation
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"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
Proteolytic acti'ation
Re&ulation of di&esti'e enzymes
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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
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>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
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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 ))
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,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
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>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
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/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
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/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
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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
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"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
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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
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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
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+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
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"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
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& & 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
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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
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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.
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Klasifikasi Enzim
Sistem Penamaan Enzim
Spesifitas Enzimacam!macam "entuk Enzim
Klasifikasi Enzim
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Klasifikasi Enzim
# kelas
$ksidoreductase
%ransferase
&idrolase
'somerase(igase
Sistem Penamaan Enzim
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Sistem Penamaan Enzim
)eaksi dan enzim yang mengkatalisisnya membentuk enam
kelas *+ hingga ,- subkelas
/ bagian : 0ama pertama substratnya
0ama kedua tipe reaksi yang dikatalisis.
'nformasi tambahan1 dituliskan dalam tanda kurung di bagian
akhir2
misalnya enzim yang mengkatalisis reaksi (!malat 3 045
3
6 piruvat3 C$/ 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
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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
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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
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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
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&'A<* D