Enzymes PDF

Enzymes Prepa red by Dr. / Ema n Moha med lecturer. of Biochemistry Enzy mes Enzymes are protein cata lysts produced from living cells to increase t he rate of chemica l reactions (not initiate) - Ta king place in a ll living systems wit hout changing t hemselves, so t hey ca...

Enzymes Prepa red by Dr. / Ema n Moha med lecturer. of Biochemistry Enzy mes Enzymes are protein cata lysts produced from living cells to increase t he rate of chemica l reactions (not initiate) - Ta king place in a ll living systems wit hout changing t hemselves, so t hey ca lled organic biocata lysts EN=In & ZYME=Yeast  Difference between prost hetic group a nd coenzyme Coenzyme Prosthetic group Loosely attached or in free Firmly attached to protein state Ca nnot be dia lyzed or Easily dia lyzed a nd sepa rated sepa rated without destruction of enzyme It contacts enzyme only at the Always attached to protein moment of reaction fraction Always orga nic Usua lly one of vita min B inorga nic complex Cu in tyrosinase Zn in ca rbonic a nhydrase Iron porphyrin in cata lase Nature of Enzy me T hey a re protein in nature. T hey a re of two types: 1.Simple enzymes made up of only protein molecules a nd not bound to a ny non- protein groups. Nature of Enzyme 1. Holoenzymes made up of protein molecules a nd bound to non- protein components. T he protein molecule of t his holoenzyme is ca lled a poenzyme. T he non- protein component of t his holoenzyme is ca lled cofactor. 1.If cofactor is orga nic so it is ca lled coenzyme. 2. If cofactor is inorga nic so it is ca lled prost hetic group. Nature of Enzyme Active site or centre: Enzyme molecule has specia l pocket ca lled active site or centre which has two regions: 1- Binding site in which the substrate bind to the enzyme forming enzyme- substrate (ES) complex. 2- Cata lytic site in which ES complex converted to enzyme- product (EP) complex a nd subsequently dissociated to enzyme a nd products. Nature of Enzyme Active site or cent re: Each enzyme posses one or more active cent re. T he active site of enzyme contain free hydroxyl group of serine, phenolic group of tyrosine, sulf hydryl group of cysteine or imidazole group of histidine to interact wit h subst rate. Difference between Enzyme a nd inorga nic cata lysts Inorga nic cata lyst Enzyme They differ in chemica l All a re protein in nature structure Thermola bile Thermosta ble Specif ic in their reactions Non specif ic in their reactions Some enzymes need activator Don not need activator Activity is due to certain group Activity is due to whole system They need little time They need extra- time Nomenclature of Enzy me 1. Usua lly enzyme na med by adding –ase to the na me of substrate. e. g. urease, ma ltase, lactase …….. etc 2. T he enzyme na med according to type of reaction. e. g. oxidase for oxidation, reductase for reduction, hydrolase for hydrolysis. 3. O ld traditiona l na mes a re used. e. g. pepsin, trypsin. 4- composed of two words; one for its substrate, and the other for the chemical reaction catalyzed as succinate dehydrogenase, Pyruvate decarboxylase and glutamine synthase.  Enzyme nomenclature 5- Enzyme code (EC): Enzyme Commission number each enzyme has numerica l code which composed of four digits sepa rated by dots: a- T he f irst digit denotes t he class (reaction type) of enzyme. b- T he second digit denotes t he functiona l group upon which t he enzyme acts. c- T he t hird digit denotes t he coenzymes. d- T he fourt h digit denotes t he subst rates. For exa mple 1. 1. 1. 1enzyme 1mea ns oxido- reductase, 1. 1mea ns t hat functiona l group is hydroxyl group, 1. 1. 1mea ns NAD is t he coenzyme, a nd Mecha nism of Enzy me Action The enzy me molecule (E) f irst combines with a substrate molecule (S) to form an enzy me substrate (ES) complex which further dissociates to form product (P) and enzy me (E) back. Enzy me once dissociated from the complex is free to combine with another molecule of substrate and form product in a similar way. Q1: How ca n enzyme bind substrate? Q2: How ca n enzyme accelerate t he rate of chemica l reaction?  Mecha nism of Enzyme Action A chemica l reaction S P (where S is t he subst rate a nd P is t he product or products) will ta ke place when a certain number of S molecules at a ny given insta nt posses enoug h energy to attain a n activated condition ca lled t he “ t ra nsition state” , in which t he proba bility of ma king or brea king a chemica l bond to - The tra nsition state is the top of the energy ba rrier sepa rating the form t he product is very hig h. reacta nts a nd products. - Activation energy is def ined as the energy required to convert a ll molecules in one mole of reacting substa nce from the ground state to overcome the tra nsition state. - Enzyme a re said to reduce the magnitude of this activation energy. Enzyme specif icity 1- Absolute specif icity T he enzyme act only on one specif ic subst rate a nd never act on a ny ot her compound. Arginase act on a rginine Lactase act on lactose Urease act on urea Sucrase act on sucrose 2- Relative specif icity The enzyme act on one type of bond in compounds chemica lly related but at different rate. Sa liva ry a mylase act on α 1- 4 glycosidic bond of sta rch, glycogen a nd dextrin. Pa ncreatic lipase act on ester bond of different triacylglycerols. Enzyme specif icity 3- Streospecif icity or optica l specif icity The enzyme act only on one type of optica lly active substa nce. D- a mino acid oxidase act on D- a mino acid α- glucosidases act on α- glucosides 4- Group or structura l specif icity The enzyme act on specif ic type of bond at specif ic site a nd attached to specif ic group. Pepsin act on peptide bond between a mino group of a romatic a mino acid a nd ca rboxylic group of a nother a mino acid. Trypsin act on peptide bond between ca rboxylic group of basic a mino acid a nd a mino group of a nother a mino acid. Chymotrypsin act on peptide bond between ca rboxylic group of a romatic a mino acid a nd a mino group of other. Ca rboxypeptidase act on termina l peptide bond at ca rboxylic end of peptide chain. Aminopeptidase act on termina l peptide bond at a mino end of peptide chain. Enzyme specif icity 5- Dua l specif icity 1. Enzyme act on two different subst rates wit h production of two different products by one reaction. Xa nt hine oxidase act on hypoxa nt hine a nd xa nt hine 2. Enzyme act on one compound wit h production of one product by two different reaction. Isocit rate dehydrogenase act on isocit rate producing ketogluta rateby deca rboxylation a nd dehydrogenation.  Factors affecting Enzyme activity 1- Subst rate concent ration. 2- Enzyme concent ration. 3- End product concent ration. 4- Temperature. 5- pH. 6- T ime 7- Activators. 8- Inhibitors Factors affecting Enzyme activity 1. Subst rate concent ration T he cha racteristic sha pe of t he subst rate saturation curve for a n enzyme ca n be expressed mat hematica lly by t he Michaelis Menten equation:  V= Velocity at a given concentration of substrate (initia l reaction velocity) Vmax = Maxima l velocity possible with excess of substrate [S] = concentration of the substrate at velocity V Km = michaelis- consta nt of the enzyme for pa Relationship rticula r [S] between substrate. a nd Km Km shows the relationship between the substrate concentration a nd the velocity of the enzyme cata lyzed reaction. Ta ke the point in which 50% of the active site of the enzyme will be saturated by substrate, Vo = ½ Vmax, at 50% saturation  Km- is the concentration of the substrate at which a given enzyme yields one- ha lf its max. Km- is cha racteristic of a n enzyme a nd a pa rticula r substrate, a nd ref lects the aff inity of the enzyme for that substrate. Factors affecting Enzyme activity 2. Enzyme concent ration T he rate of t he reaction is direct ly proportiona l to enzyme concent ration at a ll subst rate concent ration till reach maximum due to depletion of subst rate. For exa mple, if t he enzyme concent ration ha lved, t he initia l rate of t he reaction (Vo) is reduced to one ha lf t hat of t he origina l. Factors affecting Enzyme activity 3. End product concentration T he accumulation of reaction products generally decrease enzy matic velocity. T he products combine with the active centre of enzy me and form a loose complex which inhibit enzy me activity. In process of digestion, the end products are continuously removed through absorption and the activity of digestive enzy me is continued. Factors affecting Enzyme activity 4. Temperature Factors affecting Enzyme activity 5. pH Factors affecting Enzyme activity 6. T ime Enzymatic reaction decreased wit h time due to: 1- Slight denaturation of enzyme. 2- Accumulation of end products. 3- Depletion of subst rate.  Factors affecting Enzyme activity 7. Activators Activators increase t he rate of enzyme reaction by different ways: (1) Remova l of inhibitory peptide. (2) Some enzyme contain –SH group required reducing agents to be activated.  Factors affecting Enzyme activity (3) Some enzymes require minera ls (meta l ions activators or meta l conjugated): 1- Meta l ion activated enzymes. (meta l ions a re loosely bind to enzymes) Amylase Cl Kinase Mg Ca rboxylase Mn Lipase Ca Cytochrome oxidase Cu 2- Meta l conjugated enzyme (meta lloenzyme). meta l ions a re tightly binds to enzymes Ca rbonic a nhydrase Zn Tyrosinase Cu Cata lase Fe Superoxide dismutase Mn Factors affecting Enzyme activity T he meta ls pa rticipate in one of t hree ways: 1- T hey bind subst rate to orient t hem for cata lysis. 2- T hrough redox reaction gain or loss of elect ron. 3- Elect rostatic sta bilization. Factors affecting Enzyme activity 8. Inhibitors: A- reversible B- non reversible A- 1) Competitive reversible inhibitors 8. Inhibitors 1) Competitive reversible inhibitors  Factors affecting Enzyme activity 8. Inhibitors A- 1) Competitive or reversible inhibitors Ma lonic acid is simila r to succinic acid so it competes wit h it at cata lytic site of succinate dehydrogenase leading to inhibition. Sulfona mide is simila r to PABA so it competes wit h it at cata lytic site of enzyme required for synt hesis of folic acid leading to inhibition. Dicuma rol is simila r to vita min K which competes wit h it at cata lytic site of enzyme (prot hrombinase) converting preprot hrombin Allopurinol used forinto t heprot hrombin t reat ment ofleading Gout. to Allopurinol Inhibits xa nt hine oxidase byits inhibition. competes wit h Uric acid precursors for t he active site of t he Statins reversible inhibition of HMG- CoA reductase, enzyme.t he rate- limiting step in cholesterol biosynt hesis.  Factors affecting Enzyme activity 8. Inhibitors A- 2) Non competitive reversible inhibitors In non- competitive inhibition, t he inhibitor binds at different site rat her t ha n t he subst rate- binding site, cha nge in conformation of t he enzyme molecules, which leads to t he reversible inactivation of t he cata lytic site. Non- competitive inhibitors bind reversibly eit her to t he free- enzyme or t he ES complex to form t he inactive complexes EI a nd ESI (Enzyme subst rate Inhibitioa n).  Factors affecting Enzyme activity 8. Inhibitors A- 2) Non competitive reversible inhibitors Factors affecting Enzyme activity 8. Inhibitors A- 2) Non competitive reversible inhibitors Lead ca n combine wit h sulf hydryl group of ferrochelatase enzyme required for insertion of iron into protoporphyrin leading to a nemia. Cya nide inhibit iron containing enzymes as cata lase, cytochrome c oxidase a nd peroxidase. Fluoride inhibit Mg requiring enzyme as enolase. Factors affecting Enzyme activity 8. Inhibitors A- 3- Uncompetitive reversible Inhibition: - binds only to ES complex at locations ot her t ha n t he cata lytic site. Subst rate binding modif ies enzyme st ructure, ma king inhibitor- binding site availa ble. Inhibition ca nnot be reversed by subst rate. In t his case a ppa rent Vmax a nd Km decreased. ra re phenomenon, one of t he few possible exa mples known being t he inhibition of a ry l sulphatase by hydrazine actors affecting Enzyme activity 8. Inhibitors B- Irreversible inhibition (enzyme poison). In t his type of inhibition, t he inhibitor ca nnot be reversed as: Antienzymes a re chemica l substa nces t hat irreversible inhibit certain enzymes as: Antipepsin a nd a ntit rypsin secreted by asca ris pa rasite inhibiting pepsin a nd t rypsin protecting asca ris from digestion. Antipepsin a nd a ntit rypsin secreted by gast ric a nd intestina l mucosa inhibiting pepsin a nd t rypsin protecting t his mucosa from digestion. SuicideAntit inhibitor hrombin secreted from endot helia l cell of blood vesselsinhibits Aspirin inhibitscyclooxygenases t hrombin t hus prevent coagulation of  Classif ication of enzymes Enzymes classif ied into six major classes: 1- Oxidoreductases. 2- Tra nsferases. 3- Hydrolases. 4- Lyases. 5- Isomerases. 6- Ligases.  1- Oxidoreductases: - Oxidoreductases a re enzymes t hat cata lyzing t he oxidation- reduction reactions a nd ca n be classif ied into: A) Oxidases B) Hydroperoxidases C) Dehydrogenases D) Oxygenases 1- Oxidoreductases: (A) Oxidases: Enzyme cata lyzes the remova l of hydrogen or electron from substrate a nd use oxygen only as hydrogen acceptor a nd form water or hydrogen peroxide. So that, there a re two types of oxidases: 1. Oxidases forming water as Cytochrome oxidase, Tyrosinase. 2. Oxidases forming hydrogen peroxide as Xa nthine oxidase. B) Hydroperoxidases: Enzyme acts on hydrogen peroxide a nd may be: 1. Cata lase Enzyme acts on two molecules of H2O2; one act as substrate a nd the other act as hydrogen donor. 2. Peroxidase Enzyme acts on one molecule of H2O2 a nd use glutathione act as hydrogen donor. 1- Oxidoreductases: (C) Dehydrogenases: Enzyme cata lyzes t he remova l of hydrogen from one subst rate to hydrogen ca rrier (not oxygen) usua lly coenzymes 1. NAD linked dehydrogenases as lactate dehydrogenase enzyme. 2. FAD linked dehydrogenases as Succinate dehydrogenase. (D) Oxygenases: Enzyme cata lyzes t he insertion of oxygen into subst rate a nd ca n be classif ied into: 1. Mono- oxygenase = Hydroxylase. Enzyme cata lyzes insertion of one atom of oxygen in t he form of hydroxyl group. e. g. phenyla la nine hydroxylase.  Classif ication of enzymes 2- Tra nsferases: Enzyme cata lyzes t he t ra nsfer of functiona l group (ot her t ha n hydrogen) from subst rate to ot her a nd ca n be classif ied into: (A) Tra nsglycosylase (B) Tra(C) Tra nsacylases nsphosphorylase Enzyme cata lyzes t ra nsfer of acyl group (R- CO) or acetyl group (C H3- CO) a nd need COA as ca rrier (D) Tra nsa minases (GOT), (GPT). (E) Tra nsmet hylases Classif ication of enzymes 3- Hydrolases: Enzymes act by splitting of certain bond by adding water a nd divided into: (A) Glycosidases: Enzymes act by splitting glycosidic bonds of different suga rs by adding water as a mylase, lactase, a nd sucrase. (B) Est rases: 4- Lyases: Enzymes act by splitting ester bond of different lipids Enzymes cata lyze t he addition or remova l of e. groups to or g. lipase. from subst rate wit hout hydrolysis, oxidation or reduction. Aldolase: It is a n enzyme t hat split a ldehyde from a lcohol as fructose 1, 6- bisphosphate a ldolase. Classif ication of enzymes 5- Isomerases: Enzyme cata lyzes t he interconversion of two isomers. (A) Aldose- ketose isomerase: cata lyze t he interconversion of a ldose to ketose as t riose isomerase enzyme. 6- Ligases: Enzyme cata lyzes t he link of two molecules using energy from ATP. EX: Ca rboxylase as pyruvate Ca rboxylase.