1 regulation of enzymic activity

7/30/2019 1 Regulation of Enzymic Activity

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Regulation of enzymic

activity.Inhibition. Activation.


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Activator groups

Activators may act on

Active site of the enzyme or onAllosteric site (allosteric

activators)


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Activation of enzymes.

One group of activators is made upof compounds affecting the activecenter region of an enzyme.

This group includes

1) substrates and

2) enzyme cofactors (metalions, coenzymes and prostheticgroups)


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Activation by metal ions

Metal ions can1) form part of the catalytic center

(ex. metals with variable valence (Fe+2) in

cytochromes electron transport).

2) the metal links with the substrate rather than

with the enzyme forming thereby a

metallosubstrate complex whichis more advantageous for the enzyme activity

(ex.Mg+2 or Mn+2 form complexes with ATP

for creatin kinase and ATPase).


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Activation by metal ions

3) metal ions facilitate the bindingof a substrate to the enzyme

active center or coenzyme to theapoenzyme by forming a kind ofbridge bonds.

4) metal ions can stabilize theconformation of apoenzyme(protein part of enzyme).


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Activation by coenzymes

The specific involvement of coenzymes(and prosthetic groups) in binding andcatalyzing the substrate explains their

activation of enzymic reactions. The majority of coenzymes are

synthesized from vitamins.

That is why we can use vitamins toincrease the metabolism of severalsubstances in the organism.


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1) Metal ions,

2) coenzymes (or prosthetic groups),

3) vitamins (as precursors of

coenzymes),4) substrates can be used in practice

as agents for activating the

enzymes.


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Allosteric activation

The number of activators

influence allosteric center

(allosteric activators).

These are different products

of metabolism in cells.


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Inhibition of enzymes.

The majority of inhibitors are divided into:reversible and irreversible inhibitors.

Inhibitors

Reversible

Competitive Noncompetitive

Irreversible


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If the enzyme restores its activity after

removal of inhibitor, it is reversible

inhibitor(otherwise this is irreversible

inhibitor).

Irreversible inhibitors are tightly

bound to enzyme (by covalentbonds), and after dialysis, the activity ofenzyme is not restored.

I ibl i hibit f h li t


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Irreversible inhibitor of cholinesterase -Diisopropyl fluorophosphate (DFP)belonging to the class of so-called nervepoisons results in the complete inhibition of theactive center of cholinesterase.

Acetylcholine is an ester of acetic acid andcholine. It is a mediator in transmission ofnervous impulses.

Cholinesterase is an enzyme that catalyzesthe hydrolysis of acetylcholine to cholineand acetic acid.

Choline esteraseAcetylcholine + H2O choline + acetic acid


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DFP reacts with the OH group of serine

residue of the active center (of

cholinesterase) .

As the result acetylcholine is accumulatedand the transmission of nerve impulses is

impaired.

Active OH + F P = O inactive enzyme O P = Oenzyme

OCH(CH3)2 HF OCH(CH3)2

OCH(CH3)2 OCH(CH3)2


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Reversible competitive inhibition

Competitive inhibition is the enzymicreaction retardation produced by bindingthe enzyme active center with an inhibitorstructurally related to the substrate andcapable of preventing the formation of anenzyme-substrate complex.

Under competitive inhibition conditions,the inhibitor and the substrate, beingstructurally related species, compete forthe active center of enzyme.

E + S ES E +I EI


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The competing compound present in

excess binds preferably to the active

center. The enzyme becomes bound

either to the substrate, or to the inhibitor.

A ternary complex ESI (enzyme-

substrate-inhibitor) is never

formed, which is a distinctive feature ofthis type of inhibition.


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The inhibition occurs as substrate-like inhibitors bind a

certain member of enzyme molecules, leading toincapability of forming an enzyme-substrate complex.

Therefore, competitive inhibitorinfluences the binding of the

substrate with the enzyme. The distinctive sign of competitive inhibition: the

removal of inhibitory blocking can

be accomplished by an excess ofthe substrate whose molecules eliminate theinhibitor from the active center of the enzyme moleculesand reactivate the latter to catalytic activity.


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Inhibition of succinatedehydrogenase is an example of

competitive inhibition. For succinate dehydrogenase (SDH),

succinate is a natural substrate, while

the structurally related oxaloacetate (anintermediate in the Krebs cycle),

exhibits a competitive inhibitory action.

Succinate fumarate oxaloacetate malonate

FAD FADH2

SDH (Inhibitor) (inhibitor)


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Effect of a competitive inhibitor.


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In presence of inhibitor the rate of reaction

will decrease, but we can remove the

inhibition by an excess of substrate. E + S ES

E + I EI

EI + S ES + I That is why, the value of Vmax in case of

competitive inhibition (VmaxI), doesnt

change. But Km increases. Because this inhibitor

affects the affinity of enzyme for substrate.


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Noncompetitive inhibition of

enzymes is the retardation associatedwith the effect of an inhibitor on thecatalytic conversion rather than on the

substate-enzyme binding.

A noncompetitive inhibitor either directly

binds the catalytic groups of theenzyme active centeror, on binding withthe enzyme, leaves the active center free

and induces conformation changes in it.


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Noncompetitive inhibition peculiarity The conformational changes affect the

structure of the catalytic site andprevent its interaction with the substate.

Since the noncompetitive inhibitor exhibits

no effect on the substrate binding, in thiscase (as distinct from competitive

inhibition) formation of a ternary

complex ESI (E+S+IESI) is observed. However, no conversion of this complex to

any reaction products occurs.

Heavy metal ions and their organic


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Heavy metals act as noncompetitive inhibitors only when

taken in small concentrations. Taken in excess, they act

as inactivators, or denaturants (this denaturation).

Heavy metal ions and their organiccompounds belong to noncompetitiveinhibitors of enzymes. For this reaction, theions of heavy metals (mercury, lead, cadmium,

arsenic and some others) are very toxic. For example, they can block the S-H

groups that make part of the catalyticsite of an enzyme. We cannot remove thisinhibitor by means of the increase of substrateconcentration, only with the help of compoundscalled reactivators.

EI + reactivator E + reactivator + I


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Effect of a noncompetitive inhibitor.


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Application of inhibitors in medicine. In therapy, noncompetitive inhibitors (mercury-,

arsenic-, and bismuth-containing preparations)

are used that are capable of noncompetitivelyinhibiting enzymes in the organism cells or in thecells of pathogenic bacteria, which actuallydetermines the medical effects of thesepreparations.

A number of preparations, such as neostigminemethylsulphate, physostigmin and sevine,depress reversibly the enzyme (choline esterase(CE)). They are competitive inhibitors.

Their action is associated with accumulatedacethylcholine. But as they are reversibleinhibitors, their effect subside gradually, since themore of acethylcholine is accumulated, the faster iteliminates the inhibitor from the active center of

choline esterase.


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Sulphanilamides are used for

treatment of certain infectiousdiseases caused by bacteria.

In bacteria para-aminobenzoic acid is

used for the synthesis of folic acid (the

folic acid is the factor of growth for

bacteria).

Owing to the structural congenerity,

sulphanilamide blocks reaction of folic acid

synthesis, leading to the inhibition of

bacterial growth


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For humas folic acid is a vitamin. It is notsynthesized in the organism.

That is why, sulphanilamides do noteffect the human cells.

To create the saturation, and to eliminate p-

aminobenzoic acid from the enzyme inbacteria, first we must use the large dose

(several tablets at once). And then to

prevent metabolism of the drug and itssecretion out of the organism we must use

the usual doses.


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Application of irreversible inhibitors

The toxicity of irreversible inhibitors ofCE (the excess of acethylcholine

poduces a toxic action on the organism)

is by far superior. For this reason they are widely used

against pests, domestic vermins and

rodents (for example, chlorophos), andas warfare gases (sarin and tabun).


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Regulation of enzymic activity.

Allosteric regulation of enzymic

activity.


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The allosteric regulation is characteristiconly a special group of enzymes with

quaternary structure possessing regulatorycenters for binding allosteric effectors.

Regulation

Covalent Allosteric(with the help ofallosteric site)

a)chemical modificationb)activation of zymogens

That is why distinctive properties of allosteric


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That is why, distinctive properties of allostericenzymes are:

1) They have quarternary structure, consist of 2and above subunits.

2) They have allosteric center (some of theenzymes may possess several allostericcenters).

3) They do not have hyperbolic shape on the

graph of Michaelis-Menten.V simple enzyme

allosteric

enzyme

[S]


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They have characteristicsigmoidal curve (as in case ofhemoglobin saturation withoxygen). Because the activecenters of enzyme subunitsfunction cooperatively .

The affinity of every next activecenter for substrate is defined bythe saturation degree of thepreviously involved centers.


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Coordinated functioning of the centers

depends on allosteric effectors.

The mechanism of action of an allostericinhibitor on enzyme is effected via a changeof the enzymes active center conformation.The observed decrease of enzymic reaction rate

is due either to an increase in Km, or to adecrease in the maximal reaction rate (Vmax).

An allosteric activator, on the contrary,facilitates the conversion of the substrate inthe active center of enzyme, which isaccompanied either by a decrease in Km, or byan increase in the maximum rate Vmax.

The majority of enzymes in the cell are allosteric.They take a keyposition in metabolism.


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Allosteric enzyme can be regulated by thesystem of negative feed-back (inhibition of theinitial enzyme I the conversion chain by the endproduct).

In such a manner the first enzyme in the reactionchain is switched off as the end productconcentration increases. Such enzyme (E1) is

called heterotropic (because the substrate Aand allosteric effector (D)) are differentsubstances.

When the substrate serves as positiveeffector (allosteric activator)ABCD suchenzyme is called homotropic.

If the enzyme has both regulation it is calledhomoheterotropic.


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Covalent regulation

The activation of certain enzymes can beaccomplished via structural modifications

non in the active center. It can be:

1) the activation of an inactive precursorrefered to as proenzyme, or zymogen(Activation of zymogens).

2) the activation via addition of a specificmodifying group to the enzymemolecule (chemical modification).

A ti ti f


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Activation of zymogensExample digestive enzymes. The conversion of

inactive precursor (proenzyme, zymogen)

takes place by means of proteolysis.

S +

HCl in the stomach

autolytic pepsin action

pepsinogen pepsin

peptide, which

prevents the

interaction of S

with active

center

Trypsinogen trypsinChymotryp-sinogen

chymotrypsin(in the smallintestine)

enteropeptidase


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This is non reversible process.

For what purpose these enzymes aresynthesized firsty in inactive form?

The pancreatic production of trypsin (and ofother proteinases) in an inactive form has adefinite biological sense, since otherwise typsin,produced in its active form, could inflict adestructive proteolytic action both on thepancreatic cells and on the enzymessynthesized by pancreas (amylase, lipase and

others). These enzymes exhibit relative group substratespecificity. They are specific towards the peptidebonds and catalyze proteolysis of variousproteins and also proteins of pancreas itself.

E l f h i l difi ti )


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Example of chemical modification)

This is reversible process. This is

regulation by means of reversible

chemical modification. The chemical modification can involve

methylation, glycosylation (of course

phosphorylation) and other reactions.

Einactive Eactive(phosphorylase B) (phosphorylase A)protein kinase

H3PO4H2O

protein

reactions ofphosphorylation

and dephosphorylation.

ATPADP


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Multienzyme systems.

Each cell in the organism possesses its specific

set of enzymes. And each organell has specific

set of enzymes. That is why, each enzyme has

the definite localization in the cell, and functionsin the definite compartment of the cell. These

compartments are separated by the membranes

(for example mitochondria).

The enzyme cannot go out of this compartmentand functions their. Compartmentalization is a

distinctive feature of enzyme catalysis.


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For example, the breakdown of fatty acidstaked place in mitochondria, but theirsynthesis in cytoplasm. These processesare separated in the cell by means ofcompartmentalization of enzymes.Otherwise, the effect of conversions wouldbe equal to zero.


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In the cell, each enzyme performs its function

not independently, but rather in a closecooperation with other enzymes. Thus,

functionally interdependent individual

enzymes, compose multienzyme systems, or

complexes. There are

1) functional

2) structure-functional

3) combined types of multienzyme systemorganization.


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The functional organization is remarkable in thatthe individual enzymes are united in a function-

oriented multienzyme system through theagency of metabolites that are capable ofdiffusing from one enzyme to another.

In a functionally organized multienzyme system,

the reaction product of the first enzyme in theconversion chain serves as a substrate for thesecond enzyme. Glycolysis serves as anexample for functional organization of

multienzyme systems. All of the glycolysis enzymes persist in a state of

dissolution. Each reaction is catalyzed byindividual enzymes.


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The structure-functional organization consists inthat the enzymes form structural functon-oriented systems via enzyme-enzyme (protein-potein) interactions. In such a manner, structuralmultienzyme supermolecular complexes arebuilt up.

For example, pyruvate dehydrogenasemultienzyme complex composed of severalenzymes engaged in the oxidation of pyruvicacid or structurally related enzymes involved in acommon function, the synthesis of fatty acids.

Such multienzyme complexes are tightly boundand resist decomposition into constituentenzymes. This is their major distinction fromfunctionally organized multienzyme systems.


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Enzymes also may become fixed on thebiomembrane to form a chain. This is anexample for the mitochondrial respiratory chain

involved in energy generation and transport ofelectrons and protons. A separation of enzymesconstitutive of such systems puts an end to theiractivity.

The combined type of multienzyme systemoganization is a combination of the two abovetypes i.e. one part of the multienzyme systemhas a structural, and the other one, a functionalorganization.

The example is the multienzyme system ofKrebs cycle in which some of the enzymes areunited into a structural complex (2-oxoglutaratedehydrogenase complex), while other enzymesare functionally interrelated through metabolite

mediators.


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Practical use of enzymes. In the enzymes and isozymes have the

diagnostic importance to identify the affectedorgan. Digestive enzymes (pepsin, trypsin,etc.)are used as a substitutes for a deficient enzymein the organism.

Immobilized enzymes are used in thetechnological sysntheses of a number ofhormonal preparations, in high-sensitiveanalyses of drugs. Proteolytic enzymes (trypsinand chymotrypsin), immobilized on gauze

bandages or tampons, are used in surgery forcleansing purulent wounds and necrotic tissues.

Their action consists in enzymic degradation ofdead cell proteins discharged in purulentwounds.

1 regulation of enzymic activity

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