Intro to Enzymes!

8/18/2019 Intro to Enzymes!

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Chapter Outline

I. Introduction Enzyme

i. Biologic proteins that catalyze biochemical

reactionsii. Not consumed or changed in composition

iii. Found in all body tissue (intracellular) and is↑ in serum after cell injury


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Chapter Outline

I. Introduction Function of Enzymes

i. ydration of Carbon !io"ide (respiration)

ii. Ner#e $nduction

iii. %uscle Contraction

i#. Nutrient !egradation (!igestion)

#. &ro'th and eproduction

#i. nergy *torage and +se


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nzymes

II. General Properties andDenitions

A. Components of an Enzyme

i. Active Site

, ca#ity of an enzyme 'heresubstrates bind and undergo achemical reaction.

ii. Allosteric Site

, ca#ity other than the acti#e sitethat binds regulatory (e-ector)

molecules.


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nzymes


A. Components of an EnzymeAllostericpromoter

Allosteric

Inhiitor


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nzymesII. General Properties and

Denitions

!. "erms associated #ith enzymes

i. Sustrates

ii. Cofactorsiii. Isoenzyme

iv. Apoenzyme

v. $oloenzymes

vi. Proenzyme or %ymo&ens

vii.Allosteric enzymes

viii.Inhiitors


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nzymes



i. Sustrates

*ubstances acted upon enzymes

*pecic for each of their particular

enzyme


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nzymes

II. General Properties and Denitions


ii. Cofactors

Non protein substances added in the enzymesubstrate comple" to manifest the enzymeacti#ity

a. Coenzyme or Prosthetic &roup

•

,n organic cofactor• Nucleotide (.g. N,!/ N,!0) and 1itamins

. Activator

• ,n inorganic cofactor

•

%etal ion (.g. Cl23

/ %g44

/Cu4)


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nzymes



iii. Isoenzyme

*imilar enzymatic acti#ity but di-er inphysical/ biochemical and immunologiccharacteristics

iv. Apoenzyme

5he protein portion of the enzyme

*ubject to denaturation/ in 'hich

enzyme losses its acti#ity


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nzymes



v. $oloenzyme

,n acti#e substance formed bycombination of a co2enzyme and anapoenzyme.

vi. Proenzyme or %ymo&ens

,n inacti#e enzyme precursor

.g. Coagulation factors and digesti#e

enzymes


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nzymes

II. General Properties and Denitions

!. "erms associated #ithenzymes

vii.Allosteric enzymes. egulator of cellular

processes/ but not all enzymesare allosteric.

. *ome can be allostericpro#ided that they are composedof 'uaternary structures #itht#o or more protein chain

containin& the active sites


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nzymes

• 5'o 6inds of allosteric enzymes7 – +. $omoallostery. "his is a

cooperati#e substrate binding and

acti#ation 'herein substrate is ahomotropic e-ector.

– 5herefore the binding of substrate to

one acti#e site alters the sustrateindin& a,nity and-or catalyticactivity at other active sites on themultimeric enzyme.


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nzymes

• 5'o 6inds of allosteric enzymes7

– 8. $eteroallostery. 5his merely in#ol#es theregulation byheterotropic e-ector molecules/'hich can be positive (activation) orne&ative (inhiition).

– 5hese heterotropic e-ectors usually bind at asite other than the acti#e site.

– 5hese e-ectors can acti#ate or inhibit theacti#ity of an enzyme..


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nzymes

viii. $I!I"/*S /F E%01A"IC*EAC"I/S

• An inhibitor is any compound that reduces the velocity

of the enzyme-catalyzed reaction when present in

the reaction mixture. Penicillin irreversibly (covalently) inhibits an enzyme

involved in bacterial cell wall synthesis.

Ibuprofen and many other nonsteroidal

antiinflammatory drugs (NSA!s) are reversible

competitive inhibitors of the cyclooxygenase activity of

prostaglandin "# synthase.


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nzymes

• $nhibitors that occupy the acti#e site andpre#ent a substrate molecule from bindingto the enzyme are said to be active site2

directed (or competitive3 as they4compete4 #ith the sustrate for theactive site).

• $nhibitors that attach to other parts of the

enzyme molecule/ perhaps distorting itsshape/ are said to be non2active site2directed (or non competitive


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9$N!* OF $N$B$5O*

• +. Competitive Inhiition

• $n competiti#e inhibition/ a chemicalinhibitor competes for the acti#e site 'ith

the substrates.• 5he reaction on the acti#e sites depends

upon the a,nity of the enzyme for thesustrate and for the inhiitor.

• Often/ the enzyme has a greater a:nity forthe inhibitor than it does for the substrate.


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"amples of competiti#einhibitors

• In case of 1ethanol poisonin&3 it occurs becausemethanol is o"idized to formaldehyde and formicacid 'hich attac6 the optic ner#e causing blindness.

• Ethanol( an e"ample of competiti#e inhibitor) is

gi#en as an antidote for methanol poisoning becauseethanol competiti#ely inhibits the o"idation ofmethanol.

• $t is sho'n 'hen ethanol is o"idized in preference to

methanol.• Conse;uently/ the o"idation of methanol is slo'ed

do'n so that the to"ic by2products do not ha#e achance to accumulate.


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$nhibitors

• oncompetitive Inhiition

$t is a substance that interacts 'iththe enzyme/ but usually not at theacti#e site.

$t reacts either remote from or #eryclose to the acti#e site.


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$nhibitors

• oncompetitive Inhiition

5he net e-ect of a noncompetiti#einhibitor is to change the shape ofthe enzyme and thus the acti#e site/

5he substrate can no longer interact'ith the enzyme to gi#e a reaction.


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$nhibitors

• Noncompetiti#e $nhibition

• One good e"ample of noncompetiti#einhibitor is the nerve &ases such asdiisopropyl5uorophosphate (DFP)

• 5his inhibits the acti#e site ofacetylcholine esterase by reacting

'ith the hydro"yl group of serine toma6e an ester.


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Chapter Outline

III.Enzyme Classication andomenclature

+. /6idoreductases

7. "ransferase

8. $ydrolases

9. :yases

;. Isomerases


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Enzyme Classication andomenclature

• 5he system for classication ofenzymes that also ser#es as a basisfor assigning code numbers to them.

• 5hese code numbers/ pre"ed by C/'hich are no' 'idely in use/ contain

four elements separated by points/'ith the corresponding meaning.


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Enzyme Classication andomenclature

.C. 3.3.3.3Alcohol=AD>o6idoreductase

•

• 5he rst numer sho's to 'hich of the si"main di#isions (classes) the enzyme belongs/

• 5he second &ure indicates the subclass/

• 5he third &ure gi#es the sub2subclass/

• 5the fourth &ure is the serial number ofthe enzyme in its sub2subclass.


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N=>% NO%NC?,5+

C:ASS *EC/11EDED A1E

A!!*E?IA"ED A1E

E.CC/DE/.

SCIE"IFICA1E

3. O"idoreductase

?actatedehydrogenas

e

?! 3.3.3.8@

?2?actate N,!4o"idoreductase

8. 5ransferase

8.3 ,spartateaminotransferase

*&O5( *erum&lutamateO"aloacetate

transaminase)

8.A.3.3

?2,spartate /82o"aloglutarate,minotransferase

8.8 ,lanineaminotransferase

*&05( *erum&lutamate

0yru#atetransamina

8.A.3.8

?2,lanine/ 82o"aloglutarateamino

transferase


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N=>% NO%NC?,5+

C:ASS *EC/11EDEDA1E

A!!*E?IA"ED A1E

E.CC/DE/.

SCIE"IFICA1E

.ydrolases

,l6aline0hosphatas

e

,?0 .3..3 Ortho2phosphoric/

monoesterphosphohydrolase (al6alineoptimum)

,cid0hosphatas

e

,C0 .3..8 Ortho2phosphoric/

monoesterphosphohydrolase (acid optimum)

E2,mylase ,%* .8.3.3 3/D2 !2 &lucan/

&lucanohydrolase


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nzymes


+. /6idoreductases

Catalyze redo" reaction bet'een t'o substrates ,2 4 B G , 4 B2

.g7 !ehydrogenase (?actate !ehydrogenase)


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nzymes


7. "ransferases Catalyze the transfer of a group (0hosphate/

methyl/ etc.) bet'een t'o substrates (,2H 4 BG , 4 B2H)

.g7 5ransferase (,?5/ ,*5/ &&5) and 9inase

(C9)


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nzymes


7. "ransferases Catalyze the transfer of a group (0hosphate/

methyl/ etc.) bet'een t'o substrates (,2H 4 BG , 4 B2H)

.g7 5ransferase (,?5/ ,*5/ &&5) and 9inase

(C9)


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nzymes

III.Enzyme Classication and omenclature

8. $ydrolases Catalyze hydrolysis of #arious bonds

,B 4 8O G ,O 4 B

.g7 ,mylase (,%>)/ ?ipase (?0*)/ 0hosphatase (,?0/ ,C0)

4


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nzymes


9. :yases Catalyze the remo#al of groups from

substrates 'ithout hydrolysisI the productremain double bonds

,50 G c,%0 4 00i

Fructose biphosphate aldolase (,?*)


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nzymes


9. :yases Catalyze the remo#al of groups from

substrates 'ithout hydrolysisI the productremain double bonds

,50 G c,%0 4 00i

Fructose biphosphate aldolase (,?*)


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E%01E 1EC$AIS1S

• 3. :o#erin& the activation ener&y. $t is doneby creating an en#ironment in 'hich thetransition state is stabilized

• E6ample is the strainin& the shape of asustrateJby binding the transition2stateconformation of the substrateKproductmolecules/

• 5he enzyme distorts the bound substrate(s) intotheir transition state form3

• 5hereby reducin& the amount of ener&yre'uired to complete the transition).


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?OL$N& ,C5$1,5$ONN&>

•

• 3. $ncrease the pro"imity of the reactants/

• 8. $ncrease the concentration of the reactants/

•. $ncrease the surface area of the reactants

• D. $ncrease the temperature of the reactants/

• . +se a catalyst (a substance 'hich speeds upa chemical reaction but is not used up)/

• A. +se an enzyme.


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E%01E 1EC$AIS1S

• 7. Providin& an alternativepath#ay.

• 5emporarily reacting 'ith the substrateto form an intermediate nzyme2substrate (*) comple"/ 'hich 'ould be

impossible in the absence of theenzyme.


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E%01E 1EC$AIS1S

• 83 *educin& the reaction entropychan&e.

• Bringing substrates together in the correct

orientation to react.• Considering enthalpy change (M) alone

o#erloo6s this e-ect.

• 5he entropic e-ect in#ol#esdestabilization of the ground state/ and itscontribution to catalysis is relati#ely small


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1.Lock and key

hypothesis

1/DE:S /F E%01E AC"I/

2. Induced Fit Hypothesis

. 5he change in shape is induced by the approaching substratemolecule. 5his more sophisticated model relies on the fact thatmolecules are Pe"ible because single co#alent bonds are freeto rotate.


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CHARACTERISTICS AND PROPERTIES OF ENZYMES


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nzymes

Enzyme @inetics

Catalytic %echanism of nzymesi. Asolute specicity

Combines 'ith only one substrate andcatalyzes only one reaction (.g. C9/ ?!)

ii. Group specicity

Combine 'ith all substrates containing aparticular chemical group (.g. ,C0/ ,?0)

iii. !ond specicity

*pecic to chemical bonds (.g. ,%>/ ?0*)

iv. Sterioisometric specicity

Combine 'ith one optical isomer (.g. ?!/&A0!)


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nzymes

I?.Enzyme @inetics

i. Catalytic %echanism of nzymes nzymes catalyze physiologic reactions by

lo'ering the acti#ation energy le#el thatthe reactants must reach


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nzymes

I?.Enzyme @inetics

i. Catalytic %echanism of nzymes

i. elationship bet'een nzyme/ *ubstrate

and 0roduct


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nzymes

I?.Enzyme @inetics

i. Catalytic %echanism of nzymes

i. elationship bet'een nzyme/ *ubstrate

and 0roduct


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Order of eaction

• the order of the reaction can bespecied in terms of the order #ithrespect to each specic reactantor the overall order of thereaction.

• Consider the reaction m, 4

nBQRRRS C.• 5he rate e;uation is R 6T,UmTBUn.


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Order of eaction

• Consider the reaction m, 4 nBQRRRS C.

• 5he rate e;uation is * BAm!n.

• $f the e"ponent m in the e;uation is 3/ then thereaction is said to be First order 'ith respect to ,.

• $f m R 8/ $n then/ 8, 4 3B QRRRS 3C) then it is saidto be second order 'ith respect to , and rst order

'ith respect to B.• No'/ if m R 3 and n R 3/ since it is rst order 'ith

respect to , and B/ then the o#erall order of thereactions said to be Second order (or m 4 n).


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,?F 2?$F

• $A:F2:IFE

• By denition/ alf2life (t3K8) is the time re;uiredfor half of the original concentration of thelimiting reactant to be used up as the reactionta6es place or half2life is e;ual to V.AW K9.

• 5hus/ the larger the rate constant (9)/ thefaster 'ill deplete the substrate.

•

,s noted/ in a rst order reaction/ the half2lifeis inversely proportional to the rateconstant (B).


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Chapter Outline

I?.Enzyme @inetics

ii. Factors that In5uence Enzymatic*eactions

3. *ubstrate Concentration8. nzyme Concentration

. p

D. 5emperature

. Cofactors

A. $nhibitors


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Chapter Outline

I?.Enzyme @inetics


3. *ubstrate Concentration First order Binetics (%ichaelis2 %enten

hypothesis)

eaction rate is proportional to the

substrate concentration.


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Chapter Outline

I?.Enzyme @inetics


8. nzyme Concentration %ero2order Binetics

Only a "ed number of substrate (ine"cess) is con#erted to product per

second


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Chapter Outline

I?.Enzyme @inetics


*ubstrate and nzyme Concentration First order and %ero /rder Binetics


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Chapter Outline

I?.Enzyme @inetics

ii. Factors that In5uenceEnzymatic *eactions

. p Common range @.V2X.V

Controlled by bu-ers

D. 5emperature

Lithin YV.3ZC

nzyme is acti#eat 8ZC/ VZC/

@ZC.


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nzymes *ources *ubstrates Optimum p

*ucrase $ntestine *ucrose A.8

ibonuclease 0ancreas [2[V2Cytidylyl adenine

@.V

\2&lucosidase

>east %ethyl2E2!2glucoside

.D

,cetylcholinesterase rythrocytes ,cetylcholine @.

nolase abbit %uscle 820hospho2!2&lycerate

A.X

,rginase Beef ?i#er ?2,rginine X.D2W.@

0epsin &astricmucosa

,cetyl ?20henylalanine

/ ?2phenylalanine

3.28.

Optimum pH of Different Enzymes


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Chapter OutlineI?.Enzyme @inetics


. Cofactors

,cti#ators7 %etalic (Ca84) and Non %etallic(Cl2 )

Coenzymes (prosthetic groups)7 8nd substrates (N,!)


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Chapter Outline

I?.Enzyme @inetics


A. $nhibitors


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Chapter Outline

I?.Enzyme @ineticsiii. 1easurement of Enzyme Activity %easurement of catalytic acti#ity

+. in product concentration

7. in substrate concentration

8. or in coenzyme concentration(N,!)

9. in altered enzyme concentration


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Chapter Outline

I?.Enzyme @ineticsiii. 1easurement of Enzyme Activity %easurement of catalytic acti#ity

+. in product concentration

7. in substrate concentration

8. or in coenzyme concentration(N,!)

9. in altered enzyme concentration


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Chapter Outline

I?.Enzyme @inetics

iii. 1easurement of Enzyme Activity

%easurement of catalytic acti#ity

a. !ependent on enzyme concentrationb. 0erformed in zero2order 6inetics (linear

phase)


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Chapter Outline

I?.Enzyme @inetics


&eneral methods of measuring

enzymatic reaction3. Fi"ed time (5'o point) ,ssay

8. Continuous2monitoring or 6ineticassays


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Chapter Outline

I?.Enzyme @inetics




eagents are combined and theamount of reaction is measured

(,%*/ ?0*/ ,C0/ ,?0)


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Chapter Outline

I?.Enzyme @inetics




eagents are combined and theamount of reaction is measured.


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Chapter Outline

I?.Enzyme @inetics

iv. Calculation of Enzyme Activity

+. I (EC)

,mount of enzyme that 'ill catalyze thereaction of 3 ]mol of substrate perminute (]mol Kmin)

7. @at (SI)

,mount of enzyme that 'ill catalyze thereaction of 3 mol of substrate per second(molKs)


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Chapter Outline

I?.Enzyme @inetics

v. 1easurement of Enzyme 1ass $mmunoassays

lectrophoresis

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Intro to Enzymes!