Sunteți pe pagina 1din 71

ENZIMELE constituie catalizatori biologici de natur proteic.

. ntr-o celul se conin 104 molecule de enzime n prezent se cunosc circa 2000 de enzime. Enzimele se mpart n: - enzime simple (enzime-proteine) - enzime conjugate (enzime-proteide) Enzimele compuse sunt constituite din partea proteic apoenzim i partea neproteic - cofactor.

Apoenzima i cofactorul enzimatic formeaz molecula activ de enzim numit holoenzima.

ORGANIZAREA STRUCTURAL A ENZIMELOR


Cofactorul enzimatic: - ionii metalelor - coenzimele. Coenzimele sunt: vitaminice i nemitaminice

Coenzimele vitaminice:
Tiaminice (vit.B1) Flavinice (vit.B2) Pantotenice (vit. B3) Nicotinamidice (vit.B5) Piridoxinice (vit.B6)

Folice sau pteridinice (acidul folic)


Cobamidice (vit.B12) Biotinice

Lipoce
Chinonice Carnitice

Coenzimele nevitaminice Nucleotidele Fosfaii glucidelor Metaloporfirinele Peptidele

Ionii metalelor cofactori ai enzimelor Metaloenzimele constituie din cantitatea total a lor. Se mpart n 2 grupe: - Ionii metalelor joac rol de activatori (aceste enzime pot funciona i n lipsa metalului) - Ionii metalelor joac rol de cofactor (n lipsa ionii metalelor aceste enzime sunt inactive)

Organizarea funcional a enzimelor


n structura tridimensional a enzimei sunt sectoare cu anumit funcie. Centrul activ A locul unde se leag substratul S. n centrul activ se deosebete sectorul de contact (de ancor) n care se leag substratul, i sectorul catalitic, unde are loc transformarea substratului dup fixarea lui n componena centrului activ intr i cofactorii. Centrul reglator sau alosteric - este spaial separat de centrul activ. Moleculele care se leag de acest centru difer de substrat, ns influeneaz la fixarea i transformarea S n centrul A, modificndu-i conformaia. Substanele care se leag de centrul alosteric se numesc efectori alosterici Pot fi pozitivi activatori sau negativi - inhibitori

a enzim simpl ; b enzim conjugat; c- enzim alosteric A- centrul activ; S- Substratul R- centrul alosteric sau reglator 1- sectorul catalitic; 2 sectorul de contact; 3 - cofactorul

Deosebim enzime constitutive, care sunt prezente permanent n celule i enzime induse, biosinteza crora este activat de aciunea substratului respectiv sau de ali factori. Una i aceiai enzim poate exista sub forma unei familii de molecule numite forme moleculare multiple ale enzimelor (FMME) care se deosebesc dup proprietile fizico-chimice i imunologice. Dac aceste diferene de proprieti sun determinate genetic FMME se numesc izoenzime. Izoenzimele joac rol reglatori n metabolism i faciliteaz adaptarea metabolismului din diferite esuturi la aciunea factorilor interni i externi. Izoenzimele permit de a modifica direcia reaciilor biochimice i determin viteza reaciilor directe i inverse.

Mecanismul de aciune a enzimelor


Eficiena aciunii enzimelor const n micorarea energiei de activare a reaciei pe care o catalizeaz, formnd compleci intemediari enzim substrat

Mecanismul de aciune a enzimelor


Procesul catalizei enzimatice poate fi divizat convenional n trei etape: 1. Difuzia substratului spre enzim i legarea cu centrul activ al enzimei 2. Transformarea complexului primar enzim-substrat n unul sau n cteva complexe activate 3. Desprirea produselor reaciei de centrul ctiv al enzimei i ufuzia lor n mediul ambiant

Proprieti generale ale reglrii enzimatice


Eficien foarte nalt (crete viteza reaciilor de 1010 1012 ori)
Selectivitate strict i anumit direcie de aciune

(specificitate) Accesibilitatea reglrii fine i exacte Activitate reglabil Viteza reaciilor enzimatice este direct proporional cu cantitatea enzimei Nu se consum n procesul reaciei

Reglarea activitii la nivel de enzim are loc prin:


biosinteza de novo sau prin modificarea cantitii

unor enzime, datorit modificrii vitezei cu care este sintetizat enzima


Schimbarea activitii moleculelor de enzime existente

n celula vie.

Reglarea activitii enzimelor are loc pe cteva ci: Schimbarea activitii enzimei prin aciunea asupra centrului catalitic (efectele izosterice) 2. Aciunea asupra centrelor alosterice ale enzimei 3. Transformarea (reversibil sau ireversibil) a zimogenilor activi (predecesori ai enzimelor) n enzime active.
1.

I. Mecanismul efectului izosteric


Reglarea are loc nivelul centrului catalitic sau centrului activ Reglarea activitii enzimelor poate fi realizat prin interaciunea lor cu diferite componente biologice sau compui eterogeni care de obicei sunt numite modificatori sau reglatori enzimatici. La aciunea modificatorilor asupra enzimei reacia poate fi accelerat ( se numesc activatori) sau inhibat ( se numesc inhibitori)

Activarea
Activarea enzimelor se estimeaz prin accelerarea

reaciilor biochimice dup aciunea modificatorului. n calitate de activatori pot fi: cofactorii enzimelor, substratele i ionii metalelor

Ionii metalelor sunt activatori specifici . Deseori pentru activarea unor enzime sunt necesari civa ioni ai metalelor. De exemplu pentru activarea enzimei Na+, K+ ATPaza , ce particip la transportul cationilor monovaleni prin membrana celular, sunt necesari ionii de Mg, Na i K Activarea cu ajutorul ionilor metalelor are prin cteva ci. - Fie ca ionii metalelor intr n componena centrului activ; - Fie ca faciliteaz legarea S cu centrul A al enzimei - Fie c se leag mai nti cu S apoi complexul Me S se leag mai uor cu Enzima.

Activarea
Substratul n anumite concentraii de asemenea

ndeplinete funcia de activator. La atingerea concentraiilor de saturaie a substratului activitatea enzimei nu crete. Substratul mrete stabilitatea enzimei i faciliteaz formarea conformaiei necesare a centrului activ al enzimei.

Activarea
Activitatea unor enzime poate fi realizat prin

modificarea lor fr a afecta centrul enzimatic activ. Sunt posibile cteva tipuri de astfel de modificri: activarea precursorilor inactivi proenzimei sau zimogenului Activarea prin adiionarea unei grupri specifice modificatoare la molecula de enzim Activarea prin disocierea complexului inactiv protein-enzim activa.

Inhibarea
Dup mecanismul de aciune inhibitorii enzimelor se mpart n urmtoarele tipuri: Inhibarea competitiv Inhibarea catalitic (necompetitiv) Inhibarea noncompetitiv Inhibarea prin exces de substrat

Inhibarea competitiv
Are loc n cazul cnd inhibitorul se aseamn dup structur cu substratul i concurnd cu S se leag de centrul A al enzimei, inhibnd activitatea. E+I = EI Eliminarea inhibrii poate fi realizat prin suprasaturaie cu substrat, care fiiind n exces substituie Inhibitorul din centrul A ctiv al enzimei. Acest tip de inhibarea se mai numete inhibare izosteric. n calitate de inhibitori izosterici pot fi metabiliii, acumularea crora regleaz activitatea enzimelor, precum i substanele eterogene.

Inhibarea necompetitiv sau catalitic


Inhibarea are loc n rezultatul influenei inhibitorului

asupra transformrii catalitice a S n cantrul catalitic, dar nu asupra legrii S de E. Inhibatorul necompetitiv sau se leag nemijlocit de centrul catalitic al En. sau se leag de En n afara centrului A, modificndu-i conformai i mpedicnd legarea susbtratului. Are lor formarea E+S+I =ESI. Transformarea de mai departe a acestui complex nu are loc. Eliminarea aciunii inhibitorilor necompetitivi poate avea loc numai n rezultatul aciunii substanelor, care leag inhibitorul. Aceste substane se numesc reactivatori.

Ex. Cianidele care se leag strns cu Fe III din centrul A al citocromoxidazei, blocheaz lanul respirator se deci respiraia i ca urmare celula moare. Ionii metalelor grele sunt inhibitori necompetitivi, ns n concentraii mici. ntr-o concentraie mai mare acioneaz ca ageni denaturani.

Inhibarea noncompetitiv
Are loc prin frnarea activitii enzimei n rezultatul legrii inhibitorului de complexul ES. Acest tip de inhibitor nu se leag de En n lipsa substratului. Inhibitorul poate facilita legarea S de En, apoi blocheaz transformarea mai departe a substratului.

Inhibarea prin exces de substrat


Are loc la suprasaturaia cu substrat. Acest tip de inhibare are lor n rezultatul formrii complexului ES, incapabil de transformri catalitice ulterioare. E+S+S= ES2 Eliminarea acestui tip de inhibare are loc prin micorarea concentraiei substratului.

II. Reglarea alosteric


Este caracteristic doar pentru enzimele cu structur cuaternar, care posed centre alosterice.
Efectorii care se leag de centrul alosteric pot fi activatori i inhibitori.

Inhibitorii alosterici frneaz transformarea S n centrul activ al enzimei, prin modificarea conformaiei centrului activ. Ca rezultat se micoreaz viteza reaciei enzimatice la aceeai concentraie de substrat. Adic enzima funcioneaz parial n zadar

Activatorii alostericii accelereaz reacia enzimatic,

faciliteaz transformarea substratului n centrul activ al enzimei i respectiv majorarea vitezei reaciei enzimatice. . Ca efectori alosterici pot fi: - metaboliii; - hormonii; - ionii metalelor; - Coenzimele - Substratul (n cazurri rare, cind centrul alosteric se aseamnp dup structur cu centrul activ) Ex. Enzima citrat sintetaza.

Enzimele alosterice joac un rol important n metabolismul celulei, ocup o poziie cheie n metabolism, ntruct reacioneaz fin la modificrile metabolismului i regleaz viteza trecerii substanelor prin ntregul sistem de enzime. Existena mecanismului alosteric de reglare este determinat de necesitatea evitrii acumulrii n surplus a metaboliilor.

Ex. Blocarea reaciei prin produsul final

III. Transformarea (reversibil sau ireversibil) a zimogenilor activi

(predecesori ai enzimelor) n enzime active

Ex. Creterea concentraiei ionilor liberi de Ca n citoplasm indirect, prin legarea lor cu calmodulinul, activeaz enzima.

Acest lucru se atinge prin: distrugerea legturilor covalente cu ajutorul hidrolazelor (lipaza din seminele de recin); Restabilirea grupelor bisulfidice (-amilaza n seminele gramineelor; Fosforilarea proteinchinazei pe baza ATP (fosforilaza); Asociarea subunitilor inactive (Acetil CoA carboxilaza)

Activitatea i proprietile enzimelor pot fi modificate

la legarea sau eliberarea lor din membrane Degradarea selectiv proteolitic a enzimelor cu ajutorul proteazelor de asemenea este o cale de reglare a raportului nezimelor active n celule. Inactivarea enzimelor este posibil prin legarea lor cu inhibitorii specifici de natur proteic

Viteza reacie enzimatice


Depinde de: Concentraia substratului Cantitatea enzimei pH-ului mediului Temperatur

General Properties: Regulatory EnzymesThe

biochemical pathways that you will soon be studying are composed of groups of coordinated enzymes that perform a specific metabolic process. In general, these enzyme groups are composed of many enzymes, only a few of which are regulated by the mechanisms described in this lecture. Regulatory enzymes are usually the enzymes that are the rate-limiting, or committed step, in a pathway, meaning that after this step a particular reaction

General Properties: Regulatory Enzymes

(cont)Frequently, regulatory enzymes are at or near the initial steps in a pathway, or part of a branch point or cross-over point between pathways (where a metabolite can be potentially converted into several products in different pathways). In general, a cell needs to conserve energy -therefore costly (in metabolic terms) biosynthetic reaction pathways will not be operational unless a particular metabolite is required at a given

Recall that when acting as catalysts, enzyme

mediated-reactions should be reversible. However, regulatory enzymes frequently catalyze thermodynamically irreversiblereactions, that is, a large negative free energy change (-DG) greatly favors formation of a given metabolic product rather than the reverse reaction. Thus, regulation of enzyme activity, usually at the committed step of the pathway, is critical for supplying and maintaining cellular metabolitic and energy homeostasis

Two General Mechanisms that Affect Enzyme

Activity:1) control of the overall quantities of enzyme or concentration of substrates present 2) alteration of the catalytic efficiency of the enzyme

Mecanisme de reglare enzimatic


Concentraia substratului
Inhibarea reversibil prin produsele reaciei sau alte

substane Activarea sau inhivbarea alosteric Modificarea covalent Modulator protein binding Proteolitic cleavage Cantitatea enzimei prezente

Regulation of Enzyme Concentrations


The overall synthesis and degradation of a particular enzyme, also termed its turnover number, is one way of regulating the quantity of an enzyme. The amount of an enzyme in a cell can be increased by increasing its rate of synthesis, decreasing the rate of its degradation, or both.

Regulation of Enzyme Concentrations: Induction


Induction (an increase caused by an effector

molecule)of enzyme synthesis is a common mechanism -this can manifest itself at the level of gene expression, RNA translation, and post-translational modifications. The actions of many hormones and/or growth factors on cells will ultimately lead to an increase in the expression and translation of "new" enzymes not present prior to the signal. These generalizations will be covered in more detail in Dr. Bannon's lectures.

Regulation of Enzyme Concentrations: Degradation


The degradationof proteins is constantly occuring in

the cell, yet the molecular mechanisms that determine when and which enzymes will be degraded are poorly understood. The turnover number of an enzyme can be used for general comparison with other enzymes or other enzyme systems, yet these numbers can vary from minutes to hours to days for different enzymes.

Regulation of Enzyme Concentrations: Degradation (cont)


Protein degradation by proteases is

compartmentalized in the cell in the lysosome (which is generally non-specific), or in macromolecular complexes termed proteasomes. Degradation by proteasomes is regulated by a complex pathway involving transfer of a 76 aa polypeptide, ubiquitin, to targeted proteins. Ubiquination of protein targets it for degradation by the proteasome. This pathway is highly conserved in eukaryotes, but still poorly understood

Regulation of Enzyme Concentrations: Degradation (cont)


Proteolytic degradation is an irreversible mechanism.

For examples, rapid proteolytic degradation of enzymes that were activated in response to some stimulus (for example, in a signal transduction response). This type of down-regulation allows for a transientresponse to a stimulus instead of a continual response. Establishing the links between proteasomes, ubiquination and signal transduction pathways is currently a very active research area

Zymogens: Inactive Precursor Proteins


A clinically important mechanism of controlling

enzyme activity is the case of protease enzymes involved (predominantly) in food digestion and blood clotting. Protease enzymes (enzymes that degrade proteins) like pepsin, trypsin and chymotrypsin are synthesized first as larger, inactive precursor proteins termed zymogens(specifically pepsinogen, trypsinogen, and chymotrypsinogen, respectively)

Zymogens (cont)Activation of zymogens by

proteolytic cleavage result in irreversible activation. Zymogen forms allow proteins to be transported or stored in inactive forms that can be readily converted to active forms in response to some type of cellular signal. Thus they represent a mechanism whereby the levels of an enzyme/protein can be rapidly increased (post-translationally). Other examples of zymogens include proinsulin, procollagen and many blood clotting enzymes (the latter will be discussed in the

Enzyme/Substrate CompartmentationSegregation of

metabolic processes into distinct subcellular locations like the cytosol or specialized organelles (nucleus, endoplasmic reticulum, Golgi apparatus, lysosomes, mitochondria, etc.) is another form of regulation. Enzymes associated with a given pathway frequently form organized, multi-component macromolecular complexes that perform a particular cellular process. Similarly, it follows that the substrates associated with a given pathway can also be localized to the same organelle or cytosolic location. This segregation

Enzyme Regulation by Compartmentation


Membrana plasmatic Cytosol

Sistemul de transport al aminoacizilor, Na+ - K+ ATP asa

Mitocondria

Glicoliza, glicogeneza, glicogenoloiya, calea pentozofosfat, sinteza acizilor grai, catabolismul bazelor purinice i pirimidinice, ARNt sintetaza Coclul Crebs, aminoacil Lanul transporto de electroni, fosforilarea oxidativ, oxidarea acizilor grai, siclul de sintez a ureei Sinteza AdN-ului i ARN-ului Sinteza proteinelor, sinteza steroizilor, glicolizarea, detoxificarea Hidrolazele Glicozil Transferazele, glucozo-5fosfataza, formarea membranelor plasmatice i vacuolelor secretoare Catalaza, aminoacid oxidaze, urate oxidaza

Nucleul Reticulul endoplasmatic Lizosomi Apparatul Golgi

Peroxisomi

Allosteric EnzymesAllosteric enzymes-from the Greek

allosfor "other" and stereosfor "shape" (or site) meaning "other site". These enzymes function through reversible, non-covalent binding of a regulatory metabolite at a site other than the catalytic, active site. When bound, these metabolites do not participate in catalysis directly, but lead to conformational changes in one part of an enzyme that then affect the overall conformation of the active site (causing an increase or decrease in activity, hence these metabolites are termed allosteric activators or

Allosteric ExampleFeedback Inhibition-This occurs

when an end-product of a pathway accumulates as the metabolic demand for it declines. This end-product in turn binds to the regulatory enzyme at the start of the pathway and decreases its activity -the greater the endproduct levels the greater the inhibition of enzyme activity. This can either effect the Kmor Vmaxof the enzyme reaction.

Metabolic Pathway Product/ Feedback Inhibition

Allosteric Enzymes -PropertiesAllosteric enzymes

differ from other enzymes in that they are generally larger in mass and are composed of multiple subunits containing active sites and regulatory molecule binding sites. The same principles that govern binding of a substrate to an active site are similar for an allosteric regulator molecule binding to its regulatory site.

Kinetics of Allosteric Enzymes -TermsCooperativity-in

relation to multiple subunit enzymes, changes in the conformation of one subunit leads to conformational changes in adjacent subunits. These changes occur at the tertiary and quaternary levels of protein organization and can be caused by an allosteric regulator. Homotropic regulation-when binding of one molecule to a multisubunit enzyme causes a conformational shift that affects the binding of the samemolecule to another subunit of the enzyme.Heterotropic regulation-when binding of one molecule to a multi-subunit enzyme affects the binding of a differentmolecule to this enzyme (Note: These

Allosteric Enzymes -KineticsAllosteric enzymes do exhibit

saturation kinetics at high [S], but they have a characteristic sigmoidal saturation curve rather than hyperbolic curve when vois plotted versus [S] (analogous to the oxygen saturation curves of myoglobin vs. hemoglobin). Addition of an allosteric activator (+)tends to shift the curve to a more hyperbolic profile (more like Michaelis-Menten curves), while an allosteric inhibitor ()will result in more pronounced sigmoidal curves. The sigmoidicity is thought to result from the cooperativity of structural changes between enzyme subunits (again similar to oxygen binding to hemoglobin). NOTE: A true Km cannot be determined

Vovs [S] for Allosteric Enzyme


Grafice cu activatori i inhibitori

Models of Allosteric Protein


desen

Regulation by Modulator Proteins -

CalmodulinCalmodulin is a small protein(17 kDa) that can bind up to fourcalcium ions (blue dots) in the two globular domains. Whencalciumis bound, calmodulin actsas a protein co-factor to stimulatethe activity of target regulatory kinases like phosphorylase kinase,myosin kinase, Ca-ATPase and aCa/calmodulin-dependentprotein kinase. It is the structuralconformation of Ca-calmodulinthat makes it an active co-factor

Phosphorylation/Signal

TransductionPhosphorylation of one enzyme can lead to phosphorylation of a different enzyme which in turn acts on another enzyme, and so on. An example of this type of phosphorylation cascadeis the response of a cell to cyclic AMP and its effect on glycogen metabolism. Use of a phosphorylation cascade allows a cell to respond to a signal at the cell surface and transmit the effects of that signal to intracellular enzymes (usually within the cytosol and nucleus) that modify a cellular process. This process is generically referred to as being part

Regulation of Enzyme Activity by Covalent

ModificationsAnother common regulatory mechanism is the reversible covalentmodification of an enzyme. Phosphorylation, whereby a phosphate is transferred from an activated donor (usually ATP) to an amino acid on the regulatory enyme, is the most common example of this type of regulation. Frequently this phosphorylation occurs in response to some stimulus (like a hormone or growth factor) that will either activate or inactivate target enzymes via changes in Kmor kcat.

Signaling Regulation of Glycogen Synthase and Phosphorylase

Other covalent modificiations:Prenylation,

Myristoylation, Palmitoylation: The covalent addition of hydrophobic, acyl fatty acid or isoprenoid groups to soluble proteins/enzymes can alter their intracellular location. This type of hydrophobic acylation generally causes target proteins to associate with a membrane rather than the cytosol. Thus, it represents a mechanistic and functional re-compartmentalization of the target protein/enzyme (an example of a prenylated protein is the Ras oncogene discussed in lecture 11

Allosteric and Phosphorylation Regulation -Glycogen Phosphorylas