Enzymes PDF

THE CHEMISTRY OF ENZYMES  ENZYMES Enzyme  Enzymes are usually proteins; with the exception of some RNAs that catalyze their own self-cleavage.  In general, names end with suffix “ase”  Enzymes are biological catalysts...

THE CHEMISTRY OF ENZYMES  ENZYMES Enzyme  Enzymes are usually proteins; with the exception of some RNAs that catalyze their own self-cleavage.  In general, names end with suffix “ase”  Enzymes are biological catalysts – increase the rate of a reaction – not consumed by the reaction – act repeatedly to increase the rate of reactions – Reactants also called “substrates” of enzyme - Some catalyze the reaction of only one compound. Ribbon diagram of cytochrome c oxidase, the enzyme that directly uses oxygen during respiration. Enzymes as a biological catalyst ENZYMES -Others are stereo-selective; for example, enzymes that catalyze the reactions of only L-amino acids. - Others catalyze reactions of specific types of compounds or bonds; for example, trypsin catalyzes hydrolysis of peptide bonds formed by the carboxyl groups of Lys and Arg. Trypsin catalyzes the hydrolysis of peptide bonds formed by the carboxyl group of lysine and arginine. 3  ENZYMES Enzymes can increase the rate of a reaction by a factor of 109 to 1020 over an uncatalyzed reaction. CATALYST TIME OF REACTION WITH CATALYST 1 SECOND WITHOUT 3 X 1012 YEARS CATALYST 4 ENZYMES  CLASSIFICATION OF ENZYMES Enzymes are commonly named after the reaction or reactions they catalyze. Example: lactate dehydrogenase, acid phosphatase. Enzymes are classified into six major groups according to the type of reaction catalyzed: 1. Oxidoreductases: Oxidation-reduction reactions. 6  CLASSIFICATION OF ENZYMES 2. Transferase: Group transfer reactions. 3. Hydrolase: Hydrolysis reactions. 7  CLASSIFICATION OF ENZYMES 4. Lyase: Addition of two groups to a C-C double bond, or removal of two groups to create a C-C double bond. COO - COO- CH 2 CH 2 C-COO- Aconitase C-COO- + H 2O CH HO C-H - COO COO- cis-Aconitate I socitrate 5. Isomerase :Isomerization reactions. CH2 OPO3 2- CH 2 OPO3 2- O Phosphohexose O CH2 OH isomerase H HO OH HO OH H OH OH HO H a-D- Glucose-6-phosphate a-D-Fructose-6-phosphate 6. Ligase: The joining to two molecules. Tyrosine-tRNA synthetase L-tyrosyl-tRNA + AMP + PPi ATP + L-tyrosine + t-RNA 8  WHY ENZYMES? Enzymes are necessary for life to exist – otherwise reactions would occur too slowly for a metabolizing organism Enzymes DO NOT change the equilibrium constant of a reaction (accelerates the rates of the forward and reverse reactions equally) Enzymes DO NOT alter the standard free energy change, (ΔG°) of a reaction. ΔG° = amount of energy consumed or liberated in the reaction. Enzymes DO NOT change thermodynamics (can’t make a reaction spontaneous) Enzymes DO decrease the activation energy of a reaction (ΔG°). Activation Energy is the energy required to start a reaction. Enzymes DO increase the rate of reactions that are otherwise possible by DECREASING the activation energy of a reaction WHY ENZYMES?  ENZYME TERMINOLOGY Apoenzyme: The protein part of an enzyme. Cofactor: A non-protein portion of an enzyme that is necessary for catalytic function; examples are metallic ions such as Zn2+ and Mg2+. Coenzyme: A non-protein organic molecule, frequently a B vitamin, that acts as a cofactor. Substrate: The compound or compounds whose reaction an enzyme catalyzes. Active site: The specific portion of the enzyme to which a substrate binds during reaction. Schematic diagram of the active site of an enzyme and the participating components. 11  ENZYMES Enzymes physically interact with their substrates to effect catalysis E + S ↔ ES ↔ ES*  EP ↔ E + P where… E = enzyme ES = enzyme/substrate complex ES* = enzyme/transition state complex EP = enzyme/product complex P = product  Substrates bind to the enzyme’s active site – pocket in the enzyme – Binding site = where substrate binds; area that holds substrate in place via noncovalent interactions – Catalytic site = where reaction takes place  Once product is released, enzyme is available to accept another substrate molecule. Enzyme can only work on one substrate molecule at a time and is NOT changed during the reaction.  ENZYME ACTIVITY Enzyme activity: A measure of how much a reaction rate is increased. We examine how the rate of an enzyme-catalyzed reaction is affected by: Enzyme concentration. Substrate concentration. Temperature. pH 15  ENZYME ACTIVITY The effect of enzyme concentration on the rate of an enzyme- catalyzed reaction. Substrate concentration, temperature, and pH are constant. 16  ENZYME ACTIVITY The effect of substrate concentration on the rate of an enzyme- catalyzed reaction. Enzyme concentration, temperature, and pH are constant. 17  ENZYME ACTIVITY The effect of temperature on the rate of an enzyme-catalyzed reaction. Substrate and enzyme concentrations and pH are constant. 18  ENZYME ACTIVITY The effect of pH on the rate of an enzyme-catalyzed reaction. Substrate and enzyme concentrations and temperature are constant. 19  TERMS IN ENZYME CHEMISTRY Activation: Any process that initiates or increases the activity of an enzyme. Inhibition: Any process that makes an active enzyme less active or inactive. Competitive inhibitor: A substance that binds to the active site of an enzyme thereby preventing binding of substrate. Noncompetitive inhibitor: Any substance that binds to a portion of the enzyme other than the active site and thereby inhibits the activity of the enzyme. 20  MECHANISM OF ACTION Lock-and-key model of enzyme mechanism. The enzyme is a rigid three- dimensional body. The enzyme surface contains the active site. Substrate (key) fits into a perfectly shaped space in the enzyme (lock) There is lots of similarity between the shape of the enzyme and the shape of the substrate 21  MECHANISM OF ACTION The Induced-fit model of an enzyme mechanism. The active site becomes modified to accommodate the substrate. Takes into account the flexibility of proteins A substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation); close but not perfect fit of E + S C. Change in protein configuration leads to a near perfect fit of substrate with enzyme 22  MECHANISM OF ACTION-ENZYME INHIBITION INHIBITORS Interfere with the action of an enzyme Decrease the rates of their catalysis Inhibitors are a great focus of many drug companies – want to develop compounds to prevent/control certain diseases due to an enzymatic activity e.g. AIDS and HIV protease inhibitors HIV protease essential for processing of proteins in virus. Without these proteins, viable viruses cannot be released to cause further infection  MECHANISM OF ACTION-ENZYME INHIBITION INHIBITORS -Inhibitors can be REVERSIBLE or IRREVERSIBLE Irreversible Inhibitors - Enzyme is COVALENTLY modified after interaction with inhibitor-permanent inhibition. - Derivatized enzyme is NO longer a catalyst – loses enzymatic activity - Original activity cannot be regenerated - Must wait for more enzyme to be made. - Also called SUICIDE INHIBITORS e.g. Aspirin! Acetylates Ser in active site of cyclooxygenase (COX) enzyme MECHANISM OF ACTION-ENZYME INHIBITION  MECHANISM OF ACTION-ENZYME INHIBITION Reversible Inhibitors Bind to enzyme and are subsequently released Leave enzyme in original condition Three subclasses: Competitive Inhibitors Non-competitive Inhibitors Uncompetitive Inhibitors Can be distinguished by their kinetics of inhibition  MECHANISM OF ACTION-ENZYME INHIBITION Mechanism of competitive inhibition.  When a competitive inhibitor enters the active site, the substrate cannot enter.  Shape and structure of inhibitor is very similar to substrate  Inhibitor mimic substrate (or transition state) and fits into the active site  Physically blocks substrate’s access into the active site 27  MECHANISM OF ACTION-ENZYME Mechanism of competitive inhibition. INHIBITION MECHANISM OF ACTION-ENZYME INHIBITION  Mechanism of Action-ENZYME INHIBITION Transition state analog A molecule whose shape mimics the transition state of a substrate. The proline racemase reaction. Pyrrole-2- carboxylate mimics the planar transition state of the reaction. The proline racemase reaction and a mimic for the transition state. 30  MECHANISM OF ACTION-ENZYME Transition state analogs INHIBITION  are compounds that resemble the transition state of a catalyzed reaction.  Usually do not undergo a chemical reaction and can act as enzyme inhibitors by blocking their active site. Like the actual transition state species, TS analogs bind much stronger to the enzyme than simple substrate or product analogs.  Many drugs are transition state analogs.  MECHANISM OF ACTION-ENZYME Abzyme INHIBITION An antibody that has catalytic activity because it was created using a transition state analog as an immunogen. (a) The molecule below is a transition analog for the reaction of an amino acid with pyridoxal-5’-phosphate. (b) The abzyme is then used to catalyze the reaction on the next screen. 32  MECHANISM OF ACTION-ENZYME INHIBITION Mechanism of noncompetitive inhibition. The inhibitor binds itself to a site other than the active site (allosterism), thereby changing the conformation of the active site. The substrate still binds but there is no catalysis. 33  MECHANISM OF ACTION-ENZYME INHIBITION UN-COMPETITIVE INHIBITORS - Inhibitor binds to a site other than the active site, but only when substrate is bound (Binds to ES complex) Distorts active site; prevents reaction from occurring  MECHANISM OF ACTION Enzyme kinetics in the presence and the absence of inhibitors. 35  MECHANISM OF ACTION Both the lock-and-key model and the induced-fit model emphasize the shape of the active site. However, the chemistry of the active site is the most important. Just five amino acids participate in the active site in more than 65% of the enzymes studied to date. These five are His > Cys > Asp > Arg > Glu. Four of these amino acids have either acidic or basic side chains; the fifth has a sulfhydryl group (-SH). 36  CATALYTIC POWER Enzymes provide an alternative pathway for reaction. (a)The activation energy profile for a typical reaction. (b)A comparison of the activation energy profiles for a catalyzed and uncatalyzed reactions. 37  ENZYME REGULATION Feedback control: An enzyme-regulation process where the product of a series of enzyme-catalyzed reactions inhibits an earlier reaction in the sequence. The inhibition may be competitive or noncompetitive. 38  ENZYME REGULATION Proenzyme (zymogen) An inactive form of an enzyme that must have part of its polypeptide chain hydrolyzed and removed before it becomes active. An example is trypsin, a digestive enzyme. It is synthesized and stored as trypsinogen, which has no enzyme activity. It becomes active only after a six-amino acid fragment is hydrolyzed and removed from the N-terminal end of its chain. Removal of this small fragment changes not only the primary structure but also the tertiary structure, allowing the molecule to achieve its active form. 39  ENZYME REGULATION Allosterism Enzyme regulation based on an event occurring at a place other than the active site but that creates a change in the active site. An enzyme regulated by this mechanism is called an allosteric enzyme. Allosteric enzymes often have multiple polypeptide chains. Negative modulation: Inhibition of an allosteric enzyme. Positive modulation: Stimulation of an allosteric enzyme. The allosteric effect Regulator: A substance that binds to an  Binding of the regulator to a site other allosteric enzyme. than the active site changes the shape of the active site. 40  ENZYME REGULATION Effects of binding activators and inhibitors to allosteric enzymes. The enzyme has an equilibrium between the T form and the R form. 41  ENZYME REGULATION Protein modification The process of affecting enzyme activity by covalently modifying it. The best known examples of protein modification involve phosphorylation/dephosphorylation. Example: Pyruvate kinase (PK) is the active form of the enzyme; it is inactivated by phosphorylation to pyruvate kinase phosphate (PKP). 42  ENZYME REGULATION Isoenzyme (Isozymes) An enzyme that occurs in multiple forms; each catalyzes the same reaction. Example: lactate dehydrogenase (LDH) catalyzes the oxidation of lactate to pyruvate. The enzyme is a tetramer of H and M chains. H4 is present predominately in heart muscle. M4 is present predominantly in the liver and in skeletal muscle. The isozymes of lactate dehydrogenase (LDH). The electrophoresis gel depicts the relative isozyme types found in different tissues. 43  ENZYMES USED IN MEDICINE The presence of enzyme in body fluids are in small amount and the level of enzymatic activity can easily be monitored. This information can prove extremely useful. Abnormal activity (either high or low) of particular enzymes in various body fluids signals either the onset of certain diseases or their progression. e.g  A number of enzymes are assayed (measured) during myocardial infarction to diagnose the severity of the heart attack.  In some cases, administration of an enzyme is part of therapy. After duodenal or stomach ulcer operations, for instance, patients are advised to take tablets containing digestive enzymes that are in short supply in the stomach after surgery. Such enzyme preparations contain lipases, either alone or combined with proteolytic enzymes ENZYMES USED IN MEDICINE 45