Kuliah biokimia enzim

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EnzymesEnzymesCompiled by :dr. Santoso

A.IntroductionB.Mechanisms of Enzymatic

reactionC.Kinetic of enzyme activityD.Factor affecting enzyme activityE.Regulation of enzyme activity



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What Are Enzymes?What Are Enzymes?• Most enzymes

are Proteins Proteins ((tertiary and quaternary structures)

• Act as CatalystCatalyst to accelerates a reaction

• Not permanentlyNot permanently changed in the process

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EnzymesEnzymes•Are specific

for what they will catalyzecatalyze

•Are ReusableReusable•End in –asease

-Sucrase-Sucrase-Lactase-Lactase-Maltase-Maltase



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How do enzymes Work?How do enzymes Work?

Enzymes work by weakening weakening bondsbonds which which lowers lowers activation activation energyenergy

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How do Enzymes Affect Reaction Rates?

Enzymes affect the rates of reactions by lowering the amount of energy of activation required for the reactions to begin. Therefore processes can occur in living systems at lower temperatures or energy levels than it would require for these same reactions to occur without the enzymes present.

How do Enzymes Bind to Substrates

There are two proposed methods by which enzymes bind to their substrate molecules:• Lock and Key Model• Induced-Fit Model

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Enzyme-Substrate ComplexEnzyme-Substrate ComplexThe substancesubstance (reactant) an enzymeenzyme acts on is the substratesubstrate

EnzymeSubstrate Joins

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Active SiteActive Site•A restricted regionrestricted region of an enzymeenzyme

molecule which bindsbinds to the substratesubstrate.

EnzymeSubstrate

Active Site

Lock and Key Model

enzyme

S1

S2

S2 enzyme

S1

ENZYME SUBSTRATE COMPLEX

enzyme

SUBSTRATEMOLECULES

Active site P 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 the shapeshape of an enzyme’s active site

• Induced Induced by the substrate

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Induced FitInduced Fit

• A changechange in the configurationconfiguration of an enzyme’s activeenzyme’s active site site (H+ and ionic bonds are involved).

• InducedInduced by the substratesubstrate..

Enzyme

Active Sitesubstrate

induced fit

Induced-Fit Model

Enzyme Cooperativity

Some enzymes have multiple active site. It has been observed that when one substrate molecule binds to a single active site in the inactive form or tense state of the enzyme, a configurational change occurs in the other active sites making them more receptive to other substrate molecules.



Enzyme KineticsExpression for enzyme catalyzed reaction:

E + S ES E + Pk1

k-1

k2

Michaelis-Menten Equation

Rate increase with [S]

Rate levels off as approach Vmax

More S than active sites in E

Adding S has no effect

At V0 = ½ Vmax

[S] = KM

VV00 = V = Vmaxmax[S] / K[S] / KMM + + [S][S]

• Vmax occurs when enzyme active sites are saturated with substrate

• Km (Michaelis-Menten constant) reflects affinity of enzyme for its substrate

• smaller the Km, the greater the affinity an enzyme has for its substrate



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What Affects Enzyme Activity?What Affects Enzyme Activity?

• Three factors:Three factors:

1.1. Environmental Environmental ConditionsConditions

2.2. Cofactors and Cofactors and CoenzymesCoenzymes

3.3. Enzyme InhibitorsEnzyme Inhibitors

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1. Environmental Conditions1. Environmental Conditions

1.1. ExtremeExtreme Temperature Temperature are the are the most dangerousmost dangerous• high tempshigh temps may denature denature

(unfold) (unfold) the enzyme. enzyme.

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

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

TemperatureAll enzymes have an optimum temperature at which they work best. If you observe the enzyme’s activity below the specific temperature it will steadily increase until it reaches the optimum. After the optimum temperature is reached the enzymes activity drops dramatically due to denaturing.

Depending on the species, the range of optimum activity is verybroad. Above is a comparison ofhuman enzyme activity with that ofbacteria found in hot springs andoceanic vents.

pH

All enzymes have an optimum pH at which they work best. If the pH falls below or rises above the optimum value, enzymatic activity decreases as a result of denaturing.

In the human body’s digestive tractthere are variations in pH from area to area. The stomach’s juices’ pHis around 2 (acidic), the enzyme pepsin found in the gastric juices has optimum activity at a pH of 2. The smallintestine’s juice’s pH is around 8 (basic).The enzyme trypsin found in the small intestine’s juices has optimum activity at a pH of 8.

Substrate Concentration

The concentration of substrate also has an affect on the rate of enzyme activity. If the concentration of substrate is increased while the concentration of enzyme is constant, the level of enzyme activity will increase until a point of saturation is reached. At this point there are no enzymes available to react with excess substrate and the rate of the reaction stabilizes. No matter if you continue to add substrate, the reaction rate will not increase!

Increasing Substrate Concentration

Rate of ReactionPoint of Saturation, all activesites are filled with substrate.

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2. Cofactors and Coenzymes2. Cofactors and Coenzymes

• Inorganic substances Inorganic substances (zinc, iron)(zinc, iron) and vitaminsvitamins (respectively) are sometimes need for proper enzymatic activityenzymatic activity.

• Example:Example:IronIron must be present in the

quaternary quaternary structurestructure -- hemoglobinhemoglobin in order for it to pick up oxygen.pick up oxygen.

Coenzymes are bound at the active site in order to interact with the substrate and play an essential role in the catalysed reaction.They act as carriers of a variety of chemical groups.

Most water-soluble vitamins are components of 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

3. Enzyme Inhibitors

Specific for an enzymeCan be reversible or non-reversibleCompetitive inhibitorsNon-competitive inhibitors

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Competitive inhibitorsCompetitive inhibitors

chemicals that resembleresemble an enzyme’s normal substrateenzyme’s normal substrate and competecompete with it for the active active sitesite.

EnzymeCompetitive inhibitor

Substrate

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Noncompetitive InhibitorsNoncompetitive Inhibitors

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

alters the active sitealters the active site.

Enzymeactive site altered

NoncompetitiveInhibitor

Substrate

Competitive vs. Non-competitive inhibitors



Enzyme activity is regulated 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 to regulation of protein synthesis or degradation are additional, long-term ways to regulate enzyme activity

Allosteric regulation of enzyme activity

Allosteric regulation = the activation or inhibition of an enzyme’s activity due to binding of an effector molecule at a regulatory site that is distinct from the active site of the enzyme

Allosteric regulators generally act by increasing or decreasing the enzyme’s affinity for the substrate

Allosteric regulation

Many allosterically controlled enzymse show quaternary structure

Covalent modification regulates the catalytic activity 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

Biotin

Biotin serves as a CO2 carrier and is essential for pyruvate carboxylase, participates directly in the catalytic mechanism of the enzyme (as opposed to inducing a conformational change in the enzyme that indirectly affects the activity of the enzyme).

NH

C

HN

SH

(CH2)4 NHC (CH2)4 CH

O

O

Biotin Lysineside chain

pyruvatecarboxylase

Site of CO2 attachment

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 can induce a conformational change in the enzyme that either increases, or decreases, its activity.

.

OH

++

+ +

O P O-O-

OATP ADP

Top 5 reasons why phosphorylation is used to regulate enzyme activity:

1. Phosphorylation is rapidly reversible, making it possible to quickly switch between active and inactive forms of an enzyme.

2. Phosphorylation is relatively inexpensive since it does not require the synthesis of new protein molecules.

3. Results in large ∆Grxn for the phosphorylation reaction. Phosphorylation can shift the conformational equilibrium of a protein by a factor of ≈104.

4. Phosphorylation/dephosphorylation is rapid and its timing can be adjusted to meet the physiological needs of the cell.

5. Phosphorylation effects can be rapidly amplified via a kinase cascade.

Summary: Covalent modification

1. Covalent modification allows an enzyme to be rapidly activated or inactivated

2. With covalent modification, regulation of a enzyme activity is achieved at low energy costs to the cell (i.e. regulation does not require synthesis of a new enzyme or inhibitory protein).

3. Phosphorylation is a good example of how enzymes are activated and inactivated by covalent post-translational modifications

Proteolytic activation

Such as those involved in protein digestion, blood clotting, and bone and tissue remodeling, must be kept in a completely inactive state until they are needed. These enzymes are synthesized as inactive precursors (known as zymogens or proenzymes) and activated when needed by proteolytic cleavage of a specific peptide bond in the zymogen.

.

Enzyme (inactive)

Enzyme (active)

Propeptide

Proteolytic Enzyme

Pro

pe

pti

de

Proteolytic activation

Regulation of digestive enzymes

Val (Asp) Lys Ile Val

Val (Asp) Lys Ile Val

Trypsinogen

+

Enteropeptidase

Trypsin4

4

Proteolytic activationof digestive enzymes

Digestion of proteins requires simultaneous activation of several digestive enzymes.

This is achieved by synthesizing the digestive enzymes as inactive zymogens that are activated by specific proteolysis by trypsin.

Trypsin is activated by enteropeptidase catalyzed proteolysis of a unique lysine-isoleucine peptide bond (this is the “master switch” that turns on the activation of the digestive enzymes).

Pepsinogen is converted to pepsin by autocatalytic proteolysis at pH 2

• Pepsinogen has a low amount of activity at pH 2, allowing it to cleave the peptide bind between amino acids 43 and 44 to generate pepsin, that is much more active than pepsinogen.

pepsinogen (inactive)

pepsin (active)

Secretion intostomach (pH 2)

autocatalyticcleavage ofpepsinogen afteramino acid 44

Zymogen

PepsinogenChymotrypsinogenTrypsinogenProcarboxypeptidaseProelastaseProthrombinFibrinogenFactor VIIFactor XProinsulinProcollagenProcollagenase

Active Enzyme

PepsinChymotrypsinTrypsinCarboxypeptidaseElastaseThrombinFibrinFactor VIIaFactor XaInsulinCollagenCollagenase

Function

protein digestionprotein digestionprotein digestionprotein digestionprotein digestionblood clot formationblood clot formationblood clot formationblood clot formationplasma glucose homeostasiscomponent of skin and bone remodeling processes during metamorphosis, etc.

Digestive enzymes, blood clotting enzymes, and enzymes involved in bone and tissue remodeling catalyze reactions that would be disastrous if they occurred at inappropriate times or locations.

For example, if proteolytic digestion of proteins occurred in the pancreas, they would start digesting the pancreas itself. Similarly, if blood clotting factors are activated when they aren’t needed, they will initiate blood clotting throughout the body.

So, they are synthesized as inactive zymogens and are stored in this inactive state until they are needed.

Blood clot formation - an example of zymogen activations

Damaged Surface

XII

KininogenKallikrein

XIIa

XI XIa

IX IXaVIIIa

X Xa

Va

Prothrombin Thrombin

XIIIa

Fibrinogen Fibrin

Cross-linked fibrin clot

VIIVIIa

X

Tissue factor

Trauma

Intrinsic Pathway

Extrinsic Pathway

Blood clotting is an excellent example of a proteolytic cascade designed to amplify an external signal (e.g. trauma) and evoke a rapid response (blood clot formation).

Thrombin itself is inhibited by antithrombin (a serpin). This provides the body with a mechanism to prevent random blood clot formation beyond the site of injury.

1. It can occur outside of cells, since ATP is not needed to convert the zymogen into the active form of the enzyme.

2. It is not a reversible reaction. Inactivation of the active enzyme must occur by either degradation of the enzyme or by inhibition (e.g. due to the binding of an inhibitory protein to the active enzyme).

Proteolytic cleavage differs from phosphorylation

Stimulation and inhibition by control proteins

Some enzymes have regulatory proteins that bind to them and regulate their activity. cAMP-dependent protein kinase is one examples of this type of regulation.

Enzyme(active)

Enzyme(inactive)

Inhibitory Protein

Inhibitory Protein

Trypsin (orange) bound to bovine pancreatic trypsin inhibitor (violet). His 57, Asp 102, Gly 193, and Ser 195 in the active site of trypsin are shown in green, red, cyan, and blue, respectively. Lys 15 in BPTI forms a salt bridge with Asp 189 in trypsin in the trypsin:BPTI complex. Binding of bovine pancreatic trypsin inhibitor is essentially irreversible.

Serpins - An example of inhibition by control proteins

Once trypsin is activated, we need a mechanism to turn it off when it is no longer needed. Pancreatic trypsin inhibitor is used to shut off trypsin activity

– pancreatic trypsin inhibitor binds very tightly to trypsin

– pancreatic trypsin inhibitor is a member of a class of proteins known as serine protease inhibitors (serpins).

– Serpins are polypeptides that inhibit serine proteases by binding to the active sites of these enzymes.

Elastase is inhibited by α1-antitrypsin

α 1-antitrypsin inhibits elastase, a serine protease that is responsible for remodeling collagen.

Individuals in which Glu 342 in α1-antitrypsin is replaced by a lysine secrete only 15% of the normal levels for α 1-antitrypsin, resulting in uncontrolled elastase activity and the breakdown of the alveolar walls in the lung.

Note that although serpins are tight binding inhibitors or serine proteases, they do not form covalent bonds with the serine proteases.

In other words, binding of serpins to serine proteases does not involve the formation of covalent bonds between the serpins and the serine proteases.

Summary of regulatory mechanisms

1. Allosteric regulationATP activation/CTP inhibition of ATCase sigmoidal kinetics

cAMP activation of cAMP-dependent protein kinase

2. Reversible covalent modificationPhosphorylation

Ser/Thr protein kinases, Tyr kinases, kinase cascades

3. Proteolytic activationDigestive enzyme, blood clotting factors

4. Protein activators and inhibitorsSerpins

Regulating the rates of enzyme-driven reactions

Cells use inhibitors and activators to turn off and on enzymes

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

Enzyme 1 Enzyme 2 Enzyme 3Inter-

mediateInter-

mediateXProductStart of

pathway

Presence of product inhibits enzyme 1

Feedback inhibition

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

This prevents too much product from being made

An example of Feedback inhibition

This example demonstrates how an end product can inhibit the first step in its production. Isoleucine binds to the allosteric site of threonine deaminase and prevents threonine from binding to the active site because the shape of the active site is altered. When the level of isoleucine drops in the cell’s cytoplasm, the isoleucine is removed from the allosteric site on the enzyme, the active site resumes the activated shape and the pathway is “cut back on” and isoleucine begins to be produced.

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