Transcript Enzymes
Packet #10 Active Sites ◦ A special pocket that contains amino acid side chains that are complementary to the substrate Catalytic Efficiency ◦ Enzymes catalyze reactions 103 to 106 faster than uncatalyzed reactions Lower the activation energy Work in only one direction as they will not catalyze a reverse reaction Specificity ◦ Enzymes are very specific ◦ Interacting with one, or few, specific substrates and catalyzing only one type of chemical reaction Cofactors ◦ Some enzymes associate with a nonprotein cofactor that is needed for enzymic activity… Zn2+ Fe2+ ◦ …and with organic molecules that are often derivatives of vitamins Regulation ◦ Enzyme activity can be regulated Can be activated or inhibited so that the rate of product formation responds to the needs of the cell Location within the cell ◦ Many enzymes are localized in specific organelles within the cell Allows isolation of substrate or product from other competing reactions Provides a favorable environment for the reaction Allows organization of the 1000’s of enzymes present in the cell into purposeful pathways. Free Energy ◦ The portion of a system’s energy that can perform work when temperature and pressure are uniform throughout the system. Activation Energy ◦ The energy difference between reactants and the transition state ◦ Determines how rapidly the reaction occurs at a given temperature The lower the activation energy, the faster the reaction will occur The higher the activation energy, the slower the reaction will occur Transition State ◦ Represents the highest-energy structure involved in the process of a chemical reaction A chemical reaction must have enough energy to overcome the “transition state.” Temperature Increases the kinetic motion Breaks the hydrogen bonds pH Changes the ionic charges Alters the shape If the pH becomes basic, the acidic amino acid side chains will lose H+ ions If the pH becomes acidic, the basic amino acid side chains will gain H+ ions Causes the ionic bonds, that help stabilize the tertiary structures of proteins, to break. Resulting in the denaturation of the enzyme. Inhibitors ◦ Chemicals that binds to enzyme and changes its activity Competitive Non-competitive More to come later Poisons ◦ Organo-phosphorous compounds Insecticides Bind to enzymes of the nervous system and kills the organism *Concentration of Substrate to Enzyme ◦ Discussed already in class Way of describing properties of enzymes ◦ Mathematical ◦ Graphical expression Expression of reaction rates of enzymes AB+C Please read Chapter 8 ◦ Section #4 Rate vs. Enzyme ◦ Ml substrate/ min Rate vs. pH Rate ◦ Reveals the optimum pH Rate vs. Temperature ◦ Reveals the optimum temperature Rate vs. Substrate ◦ Shows a saturation curve ◦ Most definitive curve of enzyme activity Michaelus and Menten proposed a simple model that accounts for most of the features of enzymecatalyzed reactions. In this model, the enzyme reversibly combines with its substrate to form an Enzyme-Substrate Complex that subsequently breaks down to product. Results in the regeneration of a free enzyme. E + S ↔ ES E + P S = substrate E = Enzyme ES = Enzyme-substrate complex K1, k-1, k2 = rate constants Describes how reaction velocity varies with substrate concentration ◦ Rate (Reaction Velocity) vs. Substrate Concentration V0 = Vmax [S]/ Km + [S] ◦ V0 = initial reaction velocity ◦ Vmax = maximal velocity ◦ Km = Michaelis constant = (k-1 + k2)/k1 Is the substrate concentration at which rate is one-half the maximal velocity A measure of affinity of enzyme for a substrate ◦ [S] = Substrate Concentration Assumptions (3) The concentration of substrate is greater than the concentration of enzymes Remember, only one substrate is able to bind at the active site of an enzyme at any time. The rate of formation of the enzyme-substrate complex is equal to the breakdown of the enzyme-substrate complex To either E + S E + P Recall equation from earlier slide. Initial velocity Only used in the analysis of enzyme reactions Meaning, the rate of reaction is measured as soon as enzyme and substrate are mixed Characteristics of Km Km = ½ Vmax Does not vary with the concentration of enzyme Small Km Reflects high affinity(an attraction to or liking for something) of the enzyme for substrate Why? Because a low concentration of substrate is needed to reach a velocity of ½ Vmax Large Km Reflects low affinity of the enzyme for substrate Relationship of Velocity to Enzyme Concentration ◦ Rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations Example If the enzyme concentration is halved, the initial rate of the reaction (v0) is reduced to one half that of the original Order of Reaction ◦ Recall from Chemistry Will leave the details of this conclusion out. When the reaction velocity is plotted against the substrate concentration, it is not always possible to determine when Vmax has been achieved. Due to the gradual upward slope of the hyperbolic curve at high substrate concentration. However, if 1/V0 is plotted vs 1/[S] , a straight line is obtained. This plot is known as the Lineweaver-Burke Plot Can be used to calculate Km Vmax Determines the mechanism of action of enzyme inhibitors 1/V0 = Km/Vmax[s] + 1/Vmax The intercept on the x axis ◦ -1/ Km The intercept on the y axis ◦ 1/Vmax Enzyme Inhibitors ◦ Competitive Inhibitors Resemble the substrate molecule for that specific enzyme Competes for the active site Reduces the productivity of enzymes by blocking ◦ Non Competitive Inhibitors Does not directly bond to the active site of the enzyme Binds at another location and alters the shape of the enzyme so that the active site is no longer fully functional Effect on Vmax ◦ Vmax is the same in the presence of a competitive inhibitor Effect on Km ◦ Michaelis constant, Km, is increased in the presence of a competitive inhibitor Effect of Lineweaver-Burke Plot ◦ Vmax is unchanged Effect on Vmax ◦ Vmax is decreased Cannot overcome by increasing the amount of substrate Effect on Km ◦ Michaelis constant, Km, is the same ◦ Non-competitive inhibitors do not interfere with the binding of substrate to enzyme Effect of Lineweaver-Burke Plot ◦ Vmax decreases ◦ Km is unchanged An end product inhibits an initial pathway enzyme by altering efficiency of enzyme action Competitive Inhibitor ◦ Important Information Enzyme Succinate dehydrogenase Catalyzes the oxidation of succinate to fumarate Cell Respiration ◦ Malonate Structurally similar to the substrate succinate Binds at the active site of the enzyme Results in an increase of the substrate succinate in the cell However, the probability of the active site being occupied by the substrate, instead of the inhibitor, increases Non-Competitive Inhibitors ◦ Lead poisoning Lead forms covalent bonds with the sulfhydryl side chains of cysteine in proteins The binding of the heavy metal shows non-competitive inhibition Drugs ◦ Can behave as enzyme inhibitors Lactam antibiotics Penicillin Amoxicillin Inhibit one or more enzymes of bacteria walls The regulation of the reaction velocity of enzymes is essential if the organism is to coordinate its numerous metabolic pathways ◦ The control of metabolism Results in changes an enzymes shape and function by binding to an allosteric site ◦ Specific receptor site on some part of the enzyme molecule remote from the active site Allosteric inhibitor, binds at the allosteric site, and stabilizes the inactive form of the enzyme Makes the enzyme non-functional Activator, also binds at the allosteric site, and stabilizes the active form on the enzyme Makes the enzyme functional ATP and ADP are examples Most historically ◦ Substrate + ase Sucrase Catalase Mallerase ◦ International Union Biochemistry and Molecular Biology 4 digit Nomenclature Committee Numbering System 1st Major Class of Activity Only six classes recognized 2nd Subclass Type of bond acted on 3rd Subclass Group acted upon Cofactor required 4th Serial Number Sequence order Oxidoreductases ◦ Catalyze oxidation-reduction reactions Transferases ◦ Catalyze transfer of C, N or P containing groups Hydrolases ◦ Catalyze cleavage of bonds by addition of water Lyases ◦ Catalyze cleavage of C-C, C-S and certain C-N bonds Isomerases ◦ Catalyze racemization of optical or geometric isomers ◦ Catalyze isomerization ◦ Change from one isomer to another Ligases ◦ Catalyze formation of bonds between carbon and O, S, N coupled with hydrolysis of high energy phosphates (ATP) ◦ Condensation of 2 substrates with splitting of ATP