Enzymes and Enzyme Kinetics PDF

ENZYMES AND ENZYME KINETICS Prepared by: Justin Brian Chiongson, M. Sc., RCh Department Relicardo M. of Chemistry and Physics Coloso, College of Liberal Arts, Sciences and Education...

ENZYMES AND ENZYME KINETICS Prepared by: Justin Brian Chiongson, M. Sc., RCh Department Relicardo M. of Chemistry and Physics Coloso, College of Liberal Arts, Sciences and Education Ph. University of San Agustin Iloilo City D., RCh 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  Nomenclature of enzymes 8,9 Enzymes are named mostly on their function, rather than their structure. They are either named by their type of reaction or their substrate identity Substrate- the substance involved in the reaction catalyzed by the enzyme. enzyme-naming processes: Many enzymes are named after its substrate and the suffix- ase is added such. Example: Lactase; this enzyme catalyzes the hydrolysis of lactose (the substrate). The suffix -in is still used in the early discovered enzymes (mostly digestive enzymes) such as pepsin, and trypsin. An enzyme may be named after the type of reaction it catalyzes. Example: oxidase is an enzyme that catalyzes oxidation reactions. The type of substrate may also be emphasized in the name, in addition to the type of reaction. Example: glucose oxidase Classes of ENZYMES CLASSIFICATION FUNCTION Transfer of electrons (hydride ions or H OXIDOREDUCTASES atoms) TRANSFERASES Group transfer reactions Hydrolysis reactions (transfer of functional HYDROLASES groups to water) Addition of groups to double bonds, or LYASES formation of double bonds by removal of groups Transfer of groups within molecules to ISOMERASES yield isomeric forms Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions LIGASES coupled to Nelson and Cox 2005. Lehninger Principles of Biochemistry ATP cleavage 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 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. How ENZYMES work 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. ACTIVE/INACTIVE APOENZY APOENZY APOENZY ME ME ME 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  Temperature As the temperature of an enzyme-catalyzed reaction increases, the rate of reaction also increases The reaction will reach its optimum temperature at which the enzyme exhibits its maximum catalytic activity When the temperature goes beyond this point, the enzyme will start to denature. Therefore, the enzymatic activity will rapidly decrease. brainly.in  pH Most enzymes operates on a very narrow pH range Small changes in pH can cause the denaturation of the enzyme Optimum pH is the pH at the enzyme exhibits maximum activity.  toppr.com Effect of pH on enzyme activity pH optimum of pepsin (1-2) (gastric) and trypsin (8-9) (intestinal)  Substrate Concentration As the substrate concentration increases, the enzyme reaches it maximum capability Each substrate must occupy the active site for a certain period and the products must leave before it can be used by next substrate. When enzymes are "fully booked" the incoming substrates must "wait" for their turn. At this point, the enzymes have reached its saturation point. The rate of enzymatic activity is constant under saturated conditions  Enzyme Concentration The higher the enzyme concentration, the higher the enzyme activity  ENZYME Activity The enzyme activity is best described by the model devised by Michaelis and Menten for a simple enzyme reaction with a single substrate and a single product. Michaelis - Menten model states that when the rate or velocity of the reaction is examined under varying substrate concentrations : at low substrate concentration, the reaction is first order. This implies that the velocity is dependent on the substrate concentration. At high levels of substrate concentration, the rate becomes zero order. This means that the velocity is independent of substrate concentration. If the rate or velocity of the reaction is plotted against the substrate concentration, the resulting curve is hyperbolic. This means that at high substrate concentrations the active sites of all enzyme molecules are saturated with substrate. The model also defines the values of Vmax or the maximum velocity of the reaction and the KM or Michaelis constant Michaelis Constant is simply defined as the substrate concentration at half maximal velocity (rate of reaction).  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 Michaeli Menten equation Nelson and Cox 2005. Principles of Biochemistry  teachmephysiology.com Michaelis-Menten curves of enzyme inhibitio Nelson and Cox 2005. Principles of Biochemistry  mikeblaber.org ichaelis –Menten curve for competitive inhibit  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  Enzyme Regulation There are two main reasons why an enzyme is regulated. It is a waste of energy if the cell continuously produce large amount of enzyme even when the amount of substrates is very low. Therefore, enzyme production must be "turned off" If the amount of product from the enzyme- catalyzed reaction is already more than what the cell needs, then it is a waste of energy if the enzyme continues to catalyze the reaction. The enzyme must be "turned off".  Mechanisms of Enzyme Regulation Feedback control An enzyme regulation in which the product inhibits one of the reactions in a chain of enzyme- catalyzed reactions. The final product usually inhibits the activity of the first enzyme in the chain by competitive inhibition, non-competitive inhibition or some other type of inhibition. When the concentration of the final product decreases, all of the reactions in the chain proceeds. The accumulation of the final product, however, inhibits the action of the first enzyme The final product serves as the messenger if the first enzyme needs to be shutdown or not.  Proenzyme Some enzymes are produced in their inactive form. These inactive form are called proenzyme or zymogens. In order to activate these enzymes, a small part of their polypeptide chain must be removed Proenzymes are produced rather than its active form to avoid random reactions in the body. Example, Trypsin is a protease, an enzyme that catalyzes the hydrolysis of peptide bonds. It is important in the digestion of the proteins that we eat. Its inactive precursor, trypsinogen, is produced in the pancreas. If the trypsin was produced in its full active form, it would result to the random digestion of the proteins in our body. Therefore, trypsinogen is produced and only turns into its active form when it enters the digestive tract. Trypsin is an example of a proteolytic enzyme. Proteolytic enzymes are enzymes that catalyzes the breaking of peptide bonds. Because they encourage the breaking of the tissues that produce them, they are generated in their inactive form. Most digestive and blood-clotting enzymes are proteolytic enzymes. Examples of proenzyme or zymogens : Trypsinogen, pepsinogen, procaspase, prolipase.  Allosterism This regulation happens when a substance binds to a certain part of the enzyme (but not in its active site) and changes the shape of its active site. Enzymes that are regulated through this mechanism are called allosteric enzymes The substance that attaches to the allosteric enzyme is called a regulator and the site to which it binds is called the regulatory site. A regulator may inhibit the enzyme action (negative modulation) or may stimulate the enzyme action (positive modulation). The attachment of the regulator to the enzyme is noncovalent and reversible Allosteric enzymes has two kinetic states : R form (relaxed form)- This is more active form of the enzyme T form (taut form)-This is the less active form of the enzyme  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 Schematic representation of an allosteric enzyme and its regulator Negative and Positive Allosteric Control Negative vs positive feedback opentextbc.ca