Enzymes And Enzyme Kinetics PDF

ENZYMES AND ENZYME KINETICS Prepared by: Justin Brian Chiongson, M. Sc., RCh Relicardo M. Coloso, Ph. D., RCh Department of Chemistry and Physics College of Liberal Arts, Sciences and Education University of San Agustin Iloilo City ENZYMES Biological catalysts increase...

ENZYMES AND ENZYME KINETICS Prepared by: Justin Brian Chiongson, M. Sc., RCh Relicardo M. Coloso, Ph. D., RCh Department of Chemistry and Physics College of Liberal Arts, Sciences and Education University of San Agustin Iloilo City ENZYMES Biological catalysts increase the rate of reactions by a factor of between 106 to 1012 times Most enzymes are proteins Nomenclature substrate + ase, ex. amylase, protease or ends in –in, ex. pepsin, papain Characteristics of ENZYMES 1. not used up in reactions 2. highly specific 3. chemically recognize, bind and modify substrates 4. high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds 5. can be denatured and precipitated with salts, solvents and other reagents 6. require the presence of other compounds - cofactors - before their catalytic activity can be exerted Classes of ENZYMES CLASSIFICATION FUNCTION OXIDOREDUCTASES Transfer of electrons (hydride ions or H atoms) TRANSFERASES Group transfer reactions Hydrolysis reactions (transfer of functional groups to HYDROLASES water) LYASES Addition of groups to double bonds, or formation of double bonds by removal of groups Transfer of groups within molecules to yield isomeric ISOMERASES forms Formation of C-C, C-S, C-O, and C-N bonds by LIGASES condensation reactions coupled to ATP cleavage Nelson and Cox 2005. Lehninger Principles of Biochemistry  Enzyme Cofactors Nonprotein molecules or ions required by an enzyme for catalytic activity Can either be coenzymes or inorganic ions Coenzyme: Organic cofactor that is often derived from vitamins Apoenzyme: Catalytically inactive protein formed by the removal of the cofactor from an active enzyme Apoenzyme + cofactor active enzyme Inorganic ions are metal ions, such as Mg2+, Zn2+, and Fe2+ How ENZYMES work How ENZYMES work 1.Enzymes are highly specific: they catalyze only one chemical reaction, having a specific substrate. This specificity results from an enzyme’s specific 3- dimensional shape. 2.The part of the enzyme that binds to the substrate is called the active site. The active site has a 3- dimensional shape that precisely matches the 3- dimensional shape of the molecule to be reacted, called the substrate. How ENZYMES work 3. When the substrate and enzyme bind temporarily, an enzyme-substrate complex is formed. 4. The activation energy needed for the reaction to occur is reduced. 5. After the reaction is complete, the substrate has formed a new product or products and the enzyme is released to be reused. ENZYME ACTION saylordotorg.github.io LOCK-AND-KEY MODEL The substrate molecule has a specific 3-dimensional shape that allows it to fit into the specific 3-dimensional shape of an enzyme’s active site. Both enzyme and substrate already exist in these specific 3-dimensional shapes. ENZYME ACTION khanacademy.org INDUCED FIT MODEL An interaction between the enzyme and substrate induces or changes the shape of the molecules to produce a suitable fit. ENZYME ACTION Transition state DG Enzymes lower the free energy of the transition state (ΔG‡) by stabilizing the transition state ACTIVE/INACTIVE APOENZYME APOENZYME APOENZYME COENZYME PROSTHETIC GROUP Metal ion 1. A coenzyme - a non-protein organic substance which is dialyzable, thermostable and loosely attached to the protein part. 2. A prosthetic group - an organic substance which is dialyzable and thermostable which is firmly attached to the protein or apoenzyme portion. 3. A metal-ion-activator - these include K+, Fe2+, Fe3+, Cu2+, Co2+, Zn2+, Mn2+, Mg2+, Ca2+, and Mo3+. ACTIVE/INACTIVE An inactive enzyme is usually termed as ZYMOGEN or PROENZYME. These are the precursors to the active enzyme. Ex. Trypsinogen, pepsinogen, procaspase, prolipase ENZYME Activity Enzyme Activity is affected by: 1. Temperature 2. pH 3. Enzyme concentration 4. Substrate concentration 5. Covalent modification – phosphorylation or methylation 6. Inhibition 7. Allosteric effects brainly.in  toppr.com Effect of pH on enzyme activity  alevelbiology.co.uk pH optimum of pepsin (1-2) (gastric) and trypsin (8-9) (intestinal) Table 20.4 - Examples of Optimum pH for Enzyme Activity Enzyme Source Optimum pH Pepsin Gastric mucosa 1.5 β-glucosidase Almond 4.5 Sucrase Intestine 6.2 Urease Soybean 6.8 Catalase Liver 7.0 Succinate dehydrogenase Beef heart 7.6 Arginase Beef liver 9.0 Alkaline phosphatase Bone 9.5 ENZYME Activity Michaelis-Menten Equation Nelson and Cox 2005. Principles of Biochemistry Michaelis-Menten curve of enzyme reaction ENZYME Activity Lineweaver-Burk plot Double reciprocal plot, a transformation of the Michaelis- Menten equation Nelson and Cox 2005. Principles of Biochemistry  Irreversible Inhibitors Covalently bond with a specific functional group of the enzyme and render it inactive Include: – Poisons – Toxic metals – Antibiotics (penicillin and sulfa drugs) Inhibit enzymes essential to the life processes of bacteria Interfere with transpeptidase in penicillin Transpeptidase - Enzyme that is important in bacterial cell wall construction  teachmephysiology.com Michaelis-Menten curves of enzyme inhibition Nelson and Cox 2005. Principles of Biochemistry  mikeblaber.org Michaelis –Menten curve for competitive inhibition  Lineweaver-Burk plot Plots are intersecting at the Y-axis Nelson and Cox 2005. Principles of Biochemistry  With non competitive inhibitor Plots are intersecting at the X-axis mikeblaber.org Lineweaver-Burk Plot Nelson and Cox 2005. Principles of Biochemistry Plots are parallel  Regulation of Enzyme Activity - Activation of Zymogens Zymogens (proenzymes): Inactive precursors of enzymes Some enzymes that would degrade the internal structures of the cell are stored as inactive zymogens – Released when required – Activated at the location where the reaction occurs Involves the cleavage of one or more peptide bonds of the zymogen Example - Synthesis of trypsinogen trypsinogen + H 2O  enteropeptidase     trypsin + hexapeptide Table 20.6 - Examples of Zymogens Zymogen Active Enzyme Function Chymotrypsinogen chymotrypsin Digestion of proteins Pepsinogen pepsin Digestion of proteins Procarboxypeptidase carboxypeptidase Digestion of proteins Proelastase elastase Digestion of proteins Prothrombin thrombin Blood clotting Trypsinogen trypsin Digestion of proteins  Allosteric regulation They are generally activated by the substrate of the pathway and inhibited by the product of the pathway, thus only turning the pathway on when it is needed. This process is known as feedback inhibition. B, C - Intermediates of pathway ib.bioninja.com.au  Allosteric Regulation - Example Synthesis of isoleucine is a five-step process – Threonine deaminase (catalyzes in the first step) is subject to inhibition from isoleucine (final product) – Isoleucine is a noncompetitive inhibitor, which binds to an allosteric site and not an active site Exerts an inhibiting effect on the enzyme activity Reaction slows as its concentration increases and no excess isoleucine is produced Negative vs positive feedback opentextbc.ca