Encimas (ARA), Apuntes de Bioquímica

¡Descarga Encimas (ARA) y más Apuntes en PDF de Bioquímica solo en Docsity! Unit 3 Enzymes. Catalysis and enzyme kinetics. 3.1. Characteristics of biological catalysts. Coenzymes, cofactors, vitamins Enzyme nomenclature and classification 3.2. Enzyme catalysis. Transition state Active site Enzyme-substrate complex Factors involved in enzyme catalysis 3.3. Enzyme kinetics. Steady-state assumption and Michaelis-Menten equation Factors affecting the enzymatic activity Enzymatic inhibition • Reversible inhibition • Irreversible inhibition 3.4. Enzyme regulation.   Allosteric behaviour Covalent modification Proteolysis OUTLINE Nonprotein components required for the enzymatic activity: cofactor – Apoenzyme + cofactor = holoenzyme – Two types of cofactors: • Metal ions: Mg2+, Zn2+, Cu2+, Mn2+, ... • Coenzymes: small organic molecules synthesised from vitamins. Prosthetic groups: tightly bound coenzymes Cofactors deficiency promotes some health problems. COFACTORS, COENZYMES AND VITAMINS 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS a CHARACTERISTICS OF BIOLOGICAL CATALYSTS COFACTORS, COENZYMES AND VITAMINS lons Enzymes Cu?+ Fe?* or Fe?* k+ Mg?+* Mn?2+ Mo Ni2+ Se Zn?+ Cytochrome oxidase Cytochrome oxidase, catalase, peroxidase Pyruvate kinase Hexokinase, glucose 6-phosphatase, pyruvate kinase Arginase, ribonucleotide reductase Dinitrogenase Urease Glutathione peroxidase Carbonic anhydrase, alcohol dehydrogenase, carboxypeptidases Aand B 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS COFACTORS, COENZYMES AND VITAMINS Coenzyme Examples of chemical groups transferred Dietary precursor in mammals Biocytin CO, Biotin Coenzyme A Acyl groups Pantothenic acid and other compounds 5'-Deoxyadenosylcobalamin H atoms and alkyl groups Vitamin B,, (coenzyme B,,) Flavin adenine dinucleotide Electrons Riboflavin (vitamin B,) Lipoate Electrons and acyl groups Not required in diet Nicotinamide adenine dinucleotide Hydride ion (:H”) Nicotinic acid (niacin) Pyridoxal phosphate Amino groups Pyridoxine (vitamin B¿) Tetrahydrofolate One-carbon groups Folate Thiamine pyrophosphate Aldehydes Thiamine (vitamin B,) Note: The structures and modes of action of these coenzymes are described in Part ll. Table 6-2 Lehninger Principles of Biochemistry, Fifth Edition 0 2008 W.H. Freeman and Company Carboxipeptidase A (peptidyl-L-amino acid hydrolase) EC 3.4.17.1 Class: 3  Hydrolases. Subclass: 4  peptide bond 17  metallocarboxypeptidases. Entry number: 1 A series of four number serves to specify a particular enzyme. The numbers are preceded by the letters EC (enzyme commission). First number: class Second number: subclass (electron donors, type of substrate, etc.) Third number: characteristics of the reaction (functional groups, etc.) Fourth number: order of the individual entries ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS The conversion of S to P occurs because a fraction of the S molecules has the energy necessary to achieve a reactive condition known as the transition state (S-P intermediate) Enzymes (catalysts) work by lowering the free energy of activation related to the transition state A-B + C A….B….C A + B-C Ej. A-B + C A + B-C Transition state 3.2. ENZYME CATALYSIS Substrate binds at the active site of the enzyme through relatively weak forces (chymotrypsin) Specificity Catalytic power Active site 3.2. ENZYME CATALYSIS FACTORS INVOLVED IN ENZYME CATALYSIS • Proximity and orientation • Surface phenomena • Bounds tension • Presence of reactive groups 3.2. ENZYME CATALYSIS Proximity and orientation FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS FACTORS INVOLVED IN ENZYME CATALYSIS Bounds tension 3.2. ENZYME CATALYSIS General acid-base catalysis and covalent catalysis: protease Presence of reactive groups FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS Enolase General acid-base catalysis and metal ion catalysis FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS It is the analysis of the velocity (or rate) of a chemical reaction catalysed by an enzyme, and how the velocities can change on the basis of environmental parameters modifications. WHAT DO YOU HAVE TO KNOW? • How the rate of an enzyme-catalysed reaction can be defined in a mathematical way • Velocity units • What is the order of a reaction (first-order reaction/second order reaction? 3.3. ENZYME KINETICS Michaelis-Menten equation describes a curve known as a rectangular hyperbola The velocity of the product formation is: [ES]kv 2 [ES] depends on: the velocity of ES formation from E + S the velocity of its dissociation to regenerate E+S or to form E + P. ][][][ 211 ESkESkS[E]kdt d[ES]   STEADY-STATE ASSUMPTION AND MICHAELIS-MENTEN EQUATION E + S ES E + P k1 k-1 k2 3.3. ENZYME KINETICS C o n ce n tr at io n 0 Time Early stage ES formation Steady state [ES] is constant Steady-state Under experimental conditions [S]>>>[E]. The [ES] quickly reaches a constant value in such dynamic system, and remains constant until complete P formation: Steady State assumption 3.3. ENZYME KINETICS ][][][ ESEE T  ])[(]][[][][ 2111 ESkkSESkSEk T   KM, Michaelis constant ])[][(][][ 2111 ESkkSkSEk T   211 1 ][ ][][ ][ kkSk SEk ES T    121 /)(][ ][][ ][ kkkS SE ES T    M T KS SE ES   ][ ][][ ][ M T KS SEk v   ][ ][][2 Maximal velocity is obtained when the enzyme is saturated: [E]T=[ES] T[E]kV 2max  [ES]kv 2 MKS SV v   ][ ][max Michaelis-Menten Equation 1 21 k kk KM    Steady-state 3.3. ENZYME KINETICS ][][]][[,0 ][ 211 ESkESkSEk dt ESd   so 3.3. ENZYME KINETICS The rate of a enzymatic reactions depends on the substrate concentration Michaelis-Menten Turnover number, Kcat T cat E V k ][ max Kcat of an enzyme is a measure of its maximal catalytic activity. It represents the kinetic efficiency of the enzyme In the reaction kcat = k2 Kcat: turnover number: number of substrate molecules converted into product per enzyme molecule per unit time, when the enzyme is saturated with substrate First order velocity constant. Units: s-1 E + S ES E + P k1 k-1 k2 3.3. ENZYME KINETICS 3.3. ENZYME KINETICS Turnover number, K..; Enzyme Substrate ka 6) Catalase H)0, 40,000,000 Carbonic anhydrase HCO; 400,000 Acetylcholinesterase Acetylcholine 14,000 PB-Lactamase Benzylpenicillin 2,000 Fumarase Fumarate 800 RecA protein (an ATPase) ATP 0.5 Table 6-7 Lehninger Principles of Biochemistry, Fifth Edition 0 2008 W.H. Freeman and Company Factors affecting the enzymatic activity Enzyme concentration -Enzymatic activity international unit (U): quantity of enzyme able to transform 1.0 mol substrate per minute at 25ºC (under optimal conditions) - Specific enzymatic activity (U/mg): number of enzymatic unit per mg of purified protein. It indicates how pure the enzyme is. Balls: they represent proteins Red balls: enzyme molecules Both cylinders: same activity units Right cylinder shows higher specific activity than the left cylinder 3.3. ENZYME KINETICS Temperature The rates of enzyme-catalysed reactions generally increase with increasing temperature. However, at high temperatures the activity declines because of the thermal denaturation of the protein structure. pH Enzymes in general are active only over a limited pH range, and most have a particular pH at which their catalytic activity is optimal. pH changes can modify side chain, prosthetic groups and substrate charges, and consequently, the activity of the enzyme. Factors affecting the enzymatic activity 3.3. ENZYME KINETICS Enzymatic inhibition • Inhibition: velocity of an enzymatic reaction is decreased or inhibited by some agent (inhibitors) – Irreversible • Inhibitor causes stable, covalent alterations in the enzyme – Examples: » Ampicillin: causes covalent modification of a transpeptidase catalysing the synthesis of the bacterial cellular wall » Aspirin: causes covalent modification in a cyclooxygenase involved in inflammation – Reversible • Inhibitor interact with the enzyme through noncovalent association/dissociation reactions. 3.3. ENZYME KINETICS IK I ][ 1 ][ ][ SK SV v m    ][ ]][[ EI IE K I  Noncompetitive inhibition  Inhibitor interacts with both E and ES. The inhibition is not blocked when the substrate concentration increases. Vapp decreases and Km is unaffected   II KK ][ ]][[ EI IE K I   V Vapp  ][ ][ 1 ][ 1 ][ S K I K I K SV v II m               REVERSIBLE INHIBITION 3.3. ENZYME KINETICS mK 1  mK 1  Noncompetitive inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS IK I   ][ 1 ][ ][ S K SV v m     ][ ]][[ ESI IES K I   Inhibitor only combines with ES It does not bind in the active site. Vapp and Kmapp decrease ][ ][ 1 ][ S K I K SV v I m             V Vapp   m appm K K  Uncompetitive inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS Chymotrypsin inhibition by diisopropylfluorophosphate (DIFP) Ciclooxigenase inhibition by aspirin IRREVERSIBLE INHIBITION 3.3. ENZYME KINETICS Living systems must regulate the enzymatic catalytic activity to: - Coordinate metabolic processes - Promote adaptations to environmental changes - Growth and complete the living cycle in the correct way Two mechanisms of regulation: 1.- Control of the enzyme availability 2.- Control of the enzymatic activity, by means of modifications of the conformation or structure 3.4. ENZYME REGULATION Allosteric enzyme: Oligomeric organization (more than one active site and more than one effector-binding site) The regulatory effects exerted on the enzyme’s activity are achieved by conformational changes occurring in the protein when effector metabolites bind Conformational states for a protein (monomer): Taut state (T): Low substrate affinity Relaxed state (R) : High substrate affinity ALLOSTERIC REGULATION 3.4. ENZYME REGULATION Aspartate carbamoyltransferase: allosteric enzyme As product accumulates, the rate of the enzymatic reaction decreases (negative effect) Feedback inhibition3.4. ENZYME REGULATION Aspartate carbamoyltransferase: allosteric enzyme (a) ATCase: Y stale Figure12:12b Fundamentals ol Biochemistry 2/e 3.4. ENZYME REGULATION COVALENT MODIFICATION Covalent modification (target residues) Phosphorylation (Tyr, Ser, Thr, His) ATP ADP o Enz Enz Lo P e- Adenylylation (Tyr) ATP PP; o Enz Enz mel —O—CH2 ¿- H OH 0H Acetylation (Lys, «amino (amino terminus)) Acetyl-CoA HS-CoA o Enz Enz Al —CH; Myristoylation (w-amino (amino terminus)) Myristoyl-CoA HS-CoA o Enz Enz La —CH, Ubiquitination (Lys) E a Hs- É2) O-< o7 activation (Q)<-s Activated ubiquitin — HS- ne — 0 nz + L0 i c—s Ed) Activated ubiquitin ADP-ribosylation (Arg, Gin, Cys, diphthamide—a modified His) NAD nicotinamide OH 0H Methylation (Glu) S-adenosyl- S-adenosyl- methionine homocysteine Enz Enz—CH Figure 6-35 Lehninger Principles of Biochemistry, Fifth Edition 9 2008 W.11.freeman and Company Some proteins are synthesized as inactive precursors, called zymogens or proenzymes, that acquire full activity only upon specific proteolytic cleavage of one or several of their peptide bonds  It is not energy dependent  The peptide bond cleavage is irreversible Examples  Digestive enzymes  Blood clotting  Peptidic hormone (insulin)  Collagen  Caspases: apoptosis 3.4. ENZYME REGULATION PROTEOLYSIS Trypsin cleaves the peptide bond joining Arg15 - Ile16 Chymotrypsin π is an enzymatically active form that acts upon other Chymotrypsin π molecules, excising two peptides. The end product is the mature protease Chymotrypsin α, in which the three peptide chains remain together because they are linked by two disulfide bonds PROTEOLYSIS COVALENT MODIFICATION 3.4. ENZYME REGULATION