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EnzymesEnzymesCompiled by :dr. Santoso

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A.IntroductionB. Mechanisms of Enzymatic reactionC. Kinetic of enzyme activityD.Factor affecting enzyme activity

E. Regulation of enzyme activity

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What AreEnzymes?What AreEnzymes? Most enzymes are

ProteinsProteins((tertiaryand quaternarystructures)

Act asCatalystCatalyst to

accelerates areaction

Not permanentlyNot permanently

changed in theprocess

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EnzymesEnzymes Are specific forwhat they willcatalyzecatalyze

Are ReusableReusable

End in asease--SucraseSucrase--LactaseLactase--MaltaseMaltase

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How do enzymes Work?How do enzymes Work?

Enzymes workbyweakening bondsweakening bondswhichwhichlowerslowersactivation energyactivation energy

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How doEnzymesAffect Reaction Rates?

Enzymes affect the ratesof reactions by loweringthe amount of energy ofactivation required forthe reactions to begin.

Therefore processes canoccur in living systems atlower temperatures orenergy levels than itwould require for these

same reactions to occurwithout the enzymespresent.

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How do Enzymes Bind to Substrates

There are two proposed methods bywhich enzymes bind to their substrate

molecules: Lock and Key Model Induced-Fit Model

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EnzymeEnzyme--Substrate ComplexSubstrate Complex

The substancesubstance(reactant) anenzymeenzyme acts on is

the substratesubstrate

EnzymeSubstrate Joins

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Active SiteActive Site Arestricted regionrestricted region of an enzymeenzyme

molecule which bindsbinds to the substratesubstrate.

EnzymeSubstrate

Active

Site

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Lockand Key Model

enzyme

S1

S2

S2 enzymeS1

ENZYME

SUBSTRATE

COMPLEX

enzyme

SUBSTRATEMOLECULES

Active siteP P

Products

Enzyme returns from the reaction unchanged

and can now react with more substrate.

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Induced FitInduced Fit A

change in theshapeshape of an

enzymesactive

site InducedInduced by the

substrate

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Induced FitInduced Fit

A changechange in the configurationconfiguration of anenzymesactiveenzymesactive sitesite (H+and ionicbondsare involved).

InducedInduced by the substratesubstrate..

Enzyme

Active Sitesubstrate

induced fit

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Induced-Fit Model

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Enzyme Cooperativity

Some enzymes havemultiple active site. Ithas been observed thatwhen one substratemolecule binds to a

single active site in theinactive form or tensestate of the enzyme, aconfigurational changeoccurs in the other

active sites making themmore receptive to othersubstrate molecules.

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A.IntroductionB. Mechanisms of Enzymatic reactionC. Kinetic of enzyme activityD.Factor affecting enzyme activityE. Regulation of enzyme activity

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Enzyme KineticsExpression for enzyme catalyzed reaction:

E + S ES E + P

k1

k-1

k2

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Michaelis-Menten Equation

Rate increase with [S]

Rate levels off as

approach VmaxMore S than activesites in E

Adding S has no effect

At V0 = Vmax[S] = KM

VV00 == VVmaxmax[S] / K[S] / KMM + [S]+ [S]

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Vmax occurs when

enzyme active sites aresaturated withsubstrate

Km (Michaelis-Menten

constant) reflectsaffinity of enzyme forits substrate

smaller the Km, the

greater the affinity anenzyme has for itssubstrate

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WhatAffectsEnzyme Activity?WhatAffectsEnzyme Activity?

Three factors:Three factors:

1.1. Environmental ConditionsEnvironmental Conditions

2.2. Cofactorsand CoenzymesCofactorsand Coenzymes

3.3. Enzyme InhibitorsEnzyme Inhibitors

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1. Environmental Conditions1. Environmental Conditions

1.1. ExtremeExtreme TemperatureTemperature are the mostare the mostdangerousdangerous

high tempshigh temps may denature (unfold)denature (unfold)

the enzyme.enzyme.2.2. pHpH (most like 6(most like 6 -- 8 pH near neutral)8 pH near neutral)

3.3. Ionic concentrationIonic concentration (salt ions)(salt ions)

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TemperatureAll enzymes have an

optimum temperature atwhich they work best.If you observe theenzymes activity belowthe specific

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

the enzymes activitydrops dramatically dueto denaturing.

Depending on the species, therange of optimum activity is verybroad. Above isa comparison ofhuman enzyme activity with thatofbacteria found in hotspringsandoceanic vents.

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pH

A

ll enzymes have anoptimum pH at whichthey work best. Ifthe pH falls below or

rises above theoptimum value,enzymatic activitydecreases

as a result ofdenaturing.

In the human bodys digestive tractthere are variations in pH from area

toarea. The stomachs juices pHisaround 2 (acidic), the enzyme pepsinfound in the gastric juices hasoptimum activity atapHof 2. The smallintestines juicespH isaround 8 (basic).The enzyme trypsin found in the smallintestines juices hasoptimum activity atapHof 8.

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Substrate Concentration

The concentration of substrate also hasan affecton the rate

of enzyme activity. If the concentration of substrate isincreased while the concentration of enzyme is constant, thelevel of enzyme activity will increase until apointof saturationisreached. At thispoint there are no enzymesavailable toreact with excesssubstrate and the rate of the reactionstabilizes. No matter if you continue toadd substrate, thereaction rate will not increase!

Increasing Substrate Concentration

Rate of ReactionPointof Saturation,all activesitesare filled withsubstrate.

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2. Cofactorsand Coenzymes2. Cofactorsand Coenzymes

Inorganic substancesInorganic substances(zinc, iron)(zinc, iron) andvitaminsvitamins (respectively)are sometimes needforproper enzymatic activityenzymatic activity.

Example:Example:IronIron mustbe present in the quaternaryquaternarystructurestructure -- hemoglobinhemoglobin in order for it to

pick upoxygen.pick upoxygen.

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Coenzymes are bound at the active site in order to

interact with the substrate and play an essential role inthe catalysed reaction.They act as carriers of a variety of chemical groups.

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Most water-soluble vitaminsare componentsof coenzymes

Vitamin Coenzyme Deficiency

Thiamine (B1)Thiamine

pyrophosphateBeriberi (weight

loss,other problems

Riboflavin (B2) FAD+ Mouth lesions, dermatitis

Nicotinic acid(niacine)

NAD+Pellagra (dermatitis,

depression)

Pantohtinic acid Coenzyme A Hypertension

Biotin Biotin Rash, muscle pain

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3. Enzyme Inhibitors

Specific for an enzymeCan be reversible or non-reversible

Competitive inhibitorsNon-competitive inhibitors

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Competitive inhibitorsCompetitive inhibitors

chemicals thatresembleresemble anenzymes normal substrateenzymes normal substrate andcompetecompete with it for the active siteactive site.

EnzymeCompetitive inhibitor

Substrate

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Noncompetitive InhibitorsNoncompetitive Inhibitors

Inhibitors that do not enter thedo not enter the active siteactive site, butbind tobind to another partanother part of the enzymeenzyme causing theenzymeenzyme to change its shapechange its shape, which in turn

alters the active sitealters the active site.

Enzymeactive site

altered

NoncompetitiveInhibitor

Substrate

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Competitive vs. Non-competitive inhibitors

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Enzyme activity isregulated by four

different mechanisms*

(1) Allosteric control

(2) Covalent modification

(3) Proteolytic activation(4) Stimulation and inhibition by control proteins

* changes in enzyme levels due toregulation of

protein synthesisor degradation are additional,long-term ways toregulate enzyme activity

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Allosteric regulation of enzyme activity

Allosteric regulation = the activation orinhibition of an enzymesactivity due tobindingof an effector molecule ataregulatory sitethat is distinct from the active site of theenzyme

Allosteric regulators generally actby increasingor decreasing the enzymesaffinity for thesubstrate

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Allosteric regulation

Many allosterically controlled enzymse showquaternary structure

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Covalent modification regulates the catalyticactivity of some enzymes

Can either activate it or inhibit it by altering theconformation of the enzyme or by serving as a functional

group in the active site.

Enz

M if ing

gr

EnzM if inggr

Inacti Enz cti Enz

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Biotin

Biotin servesasa CO2 carrierand is essential forpyruvate

carboxylase,participates directly in the catalytic mechanism ofthe enzyme (asopposed to inducing a conformational change in theenzyme that indirectly affects the activity of the enzyme).

NH

C

HN

SH

(CH2)4 NHC (CH2)4 CH

O

O

Biotin Lysineside chain

pyruvate

carboxylase

Site of CO2 attachment

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Phosphorylation - an example of regulation by

reversible covalent modification of the enzyme

Inserting a negatively charged phosphate group

into the appropriate location in an enzyme caninduce a conformational change in the enzymethat either increases,or decreases, itsactivity.

.

OH

++

+ +

O PO-

O-

OATP ADP

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Top 5 reasons why phosphorylation is used

toregulate enzyme activity:1. Phosphorylation israpidly reversible, making itpossible to quickly

switchbetween active and inactive formsof an enzyme.

2. Phosphorylation isrelatively inexpensive since it does notrequirethe synthesisof new protein molecules.

3. Results in large Grxn for the phosphorylation reaction.Phosphorylation can shift the conformational equilibrium of aprotein by a factorof 104.

4. Ph osphorylation/dephosphorylation israpid and its timing can beadjusted to meet the physiological needsof the cell.

5. Ph osphorylation effects can be rapidly amplified viaakinasecascade.

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Summary: Covalent modification

1. Covalent modification allowsan enzyme tobe rapidlyactivated or inactivated

2. With covalent modification,regulation of a enzyme

activity isachieved at low energy costs to the cell (i.e.regulation does notrequire synthesisof a new enzyme orinhibitory protein).

3. Phosphorylation isa good example of how enzymesareactivated and inactivated by covalentpost-translationalmodifications

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Proteolytic activation

P l

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Suchas those involved in protein digestion,blood clotting,and boneand tissue remodeling, mustbe kept in a completely inactive stateuntil they are needed. These enzymesare synthesized as inactive

precursors (known aszymogensorproenzymes)and activated whenneeded by proteolytic cleavage of aspecific peptide bond in thezymogen.

.

Enz(inacti )

Enz (acti )

r ti

ProteolyticEnzyme

Pr

ti


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Regulation of digestive enzymes

Val (Asp) Lys Ile Val

Val (Asp) Lys Ile Val

Trypsinogen

+

Enteropeptidase

Trypsin4

4


of digestive enzymes

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Digestion of proteinsrequiressimultaneousactivation of several digestive enzymes.

This isachieved by synthesizing the digestiveenzymesas inactive zymogens thatare activated

by specific proteolysisby trypsin.Trypsin isactivated by enteropeptidasecatalyzed proteolysisof a unique lysine-isoleucinepeptide bond (this is the masterswitch thatturnson the activation of the digestive

enzymes).

P i i d i b

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Pepsinogen is converted topepsin byautocatalytic proteolysisatpH2

Pepsinogen hasa lowamountof activity atpH2,allowing it tocleave the peptide

bind between aminoacids 43and 44 togenerate pepsin, thatis much more activethan pepsinogen.

pepsinogen (inacti e)

pepsin(acti e)

ecretion intostomach (pH - )

autocatal ticcleavageofpepsinogenafteraminoaci

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Zymogen

PepsinogenChymotrypsinogenTrypsinogenProcarboxypeptidaseProelastaseProthrombin

FibrinogenFactorVIIFactorXProinsulinProcollagenProcollagenase

Active Enzyme

PepsinChymotrypsinTrypsinCarboxypeptidaseElastaseThrombin

FibrinFactorVIIaFactorXaInsulinCollagenCollagenase

Function

protein digestionprotein digestionprotein digestionprotein digestionprotein digestionblood clot formation

blood clot formationblood clot formationblood clot formationplasma glucosehomeostasiscomponentof skin and

bone remodelingprocesses duringmetamorphosis, etc.

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Digestive enzymes,blood clotting enzymes,and enzymesinvolved in bone and tissue remodeling catalyze reactionsthat would be disastrousif they occurred atinappropriate timesor locations.

For example, if proteolytic digestion of proteins

occurred in the pancreas, they would start digesting thepancreas itself. Similarly, if blood clotting factorsareactivated when they arent needed, they will initiateblood clotting throughout the body.

So, they are synthesized as inactive zymogensand arestored in this inactive state until they are needed.

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Blood clot formation - an example of zymogen activations

Damaged Surface

XII

KininogenKallikrein

XIIa

XI XIa

IX IXaVIIIa

X Xa

Va

ProthrombinThrombin

XIIIa

Fibrinogen Fibrin

Cross-linkedfibrin clot

VIIVIIa

X

Tissuefactor

Trauma

Intrinsic Pathway

Extrinsic Pathway

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Blood clotting isan excellent example of aproteolytic cascade designed toamplify anexternal signal (e.g. trauma)and evoke arapidresponse (blood clot formation).

Thrombin itself is inhibited by antithrombin (aserpin). Thisprovides the body witha mechanismtopreventrandom blood clot formation beyond thesite of injury.

P t l ti l diff f h h l ti

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1. It can occuroutside of cells,since ATP is notneeded to convert the zymogen into the activeform of the enzyme.

2. It is notareversible reaction. Inactivation ofthe active enzyme mustoccurby eitherdegradation of the enzyme orby inhibition (e.g.due to the binding of an inhibitory protein tothe active enzyme).

Proteolytic cleavage differs from phosphorylation

i l i d i hibi i b l i

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Stimulation and inhibition by control proteins

Some enzymes have regulatory proteins thatbind to themand regulate theiractivity. cAMP-dependentproteinkinase isone examplesof this type of regulation.

Enzyme

(active)

Enzyme

(inactive)InhibitoryProtein

InhibitoryProtein

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Trypsin (orange)bound tobovine pancreatic trypsin inhibitor(violet). His 57,Asp102, Gly 193,and Ser195 in theactive site of trypsin are shown in green,red, cyan,and blue,respectively. Lys15 in BPTI formsasaltbridge withAsp189in trypsin in the trypsin:BPTI complex. Binding of bovinepancreatic trypsin inhibitor is essentially irreversible.

Serpins - An example of inhibition by control proteins

Once trypsin isactivated,we need a mechanism to

turn itoff when it is nolonger needed. Pancreatictrypsin inhibitor is used toshutoff trypsin activity pancreatic trypsin inhibitor

binds very tightly to trypsin pancreatic trypsin inhibitor

isa memberof a classofproteinsknown as serineprotease inhibitors(serpins).

Serpinsare polypeptides

that inhibitserine proteasesby binding to the activesitesof these enzymes.

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Elastase is inhibited by 1-antitrypsin

1-antitrypsin inhibits elastase,aserine protease that isresponsibleforremodeling collagen.

Individuals in which Glu 342 in 1-antitrypsin isreplaced by a lysinesecrete only 15% of the normallevels for1-antitrypsin,resultingin uncontrolled elastase activity andthe breakdown of the alveolar wallsin the lung.

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Note thatalthoughserpins are tightbindinginhibitorsorserine proteases, they do not formcovalentbonds with the serine proteases.

In other words,binding of serpins toserineproteases does not involve the formation ofcovalentbondsbetween the serpins and theserine proteases.

S f l t h i

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Summary of regulatory mechanisms

1. Allosteric regulation

ATP activation/CTP inhibition of ATCase sigmoidal kineticscAMP activation of cAMP-dependentprotein kinase

2. Reversible covalent modificationPhosphorylation

Ser/Thrprotein kinases, Tyrkinases,kinase cascades

3. Proteolytic activation

Digestive enzyme,blood clotting factors

4. Protein activatorsand inhibitorsSerpins

Regulating the rates of enzyme driven

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Regulating the ratesof enzyme-drivenreactions

Cells use inhibitors and activators to turn off andon enzymes

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

F db k i hibiti

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Enzyme 1 Enzyme 2 Enzyme 3Inter-mediate Inter-mediateX ProductStart ofpathway

Presence of product inhibits enzyme 1

Feedback inhibition

Many enzymes are actually regulated by theend products of the reaction they catalyze

This prevents too much product from being made

An example of Feedback inhibition

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An example of Feedback inhibition

This example demonstrates how an

end product can inhibit the firststep in its production. Isoleucinebinds to the allosteric site ofthreonine deaminase and preventsthreonine from binding to the activesite because the shape of the active

site is altered. When the level ofisoleucine drops in the cellscytoplasm, the isoleucine is removedfrom the allosteric site on theenzyme, the active site resumes the

activated shape and the pathway iscut back on and isoleucine beginsto be produced.

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