Enzymes Packet #13 Chapter #8 Friday, November 23, 2018

Properties of Enzymes Catalytic Efficiency Specificity Enzymes catalyze (speed up) reactions 103 to 106 faster than uncatalyzed reactions Lower the activation energy Using the chemical equation model, enzymes 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 Friday, November 23, 2018

Properties of Enzymes II 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 Friday, November 23, 2018

Properties of Enzymes III 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. Friday, November 23, 2018

Naming Enzymes Friday, November 23, 2018

Naming of Enzymes 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 Group acted upon Cofactor required 4th Serial Number Sequence order Friday, November 23, 2018

Classes of Enzymes Friday, November 23, 2018

Classes of Enzymes I Oxidoreductases Transferases Hydrolases Lyases Catalyze oxidation-reduction reactions Transferases Catalyze transfer of C, N or P containing groups Hydrolases Catalyze cleavage of bonds by addition of water Catalyze hydrolysis reactions Lyases Catalyze cleavage of C-C, C-S and certain C-N bonds Friday, November 23, 2018

Classes of Enzymes II Isomerases Ligases Catalyze conversion of a molecule from one isomeric form to another Catalyze racemization of optical or geometric isomers Catalyze isomerization Change from one isomer to another Ligases Catalyze certain reactions in which two molecules join in a process coupled to the hydrolysis of ATP. 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 Friday, November 23, 2018

Enzyme Structure Friday, November 23, 2018

Structure Enzymes are active proteins. On the structure of an enzyme, one would find an active site where the chemical reaction takes place. Friday, November 23, 2018

Structure II Friday, November 23, 2018

Function & Specificity Friday, November 23, 2018

How Enzymes Work Friday, November 23, 2018

Gibb’s Free Energy Free Energy The portion of a system’s energy that can perform work when temperature and pressure are uniform throughout the system. Friday, November 23, 2018

Reading Energy Charts Friday, November 23, 2018

Exergonic Reaction Friday, November 23, 2018

Activation Energy Activation Energy Transition State 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.” Friday, November 23, 2018

Energy Charts Friday, November 23, 2018

Factors that Change the Shape of Enzymes Friday, November 23, 2018

Changing Shape of Enzyme I Temperature Increases the kinetic motion Breaks the hydrogen bonds Friday, November 23, 2018

Changing Shape of Enzyme I 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. Friday, November 23, 2018

Inhibiting Enzyme Function Friday, November 23, 2018

Inhibiting Enzyme Function Inhibitors Chemicals that binds to enzyme and changes its activity Competitive Inhibitor that binds at the active site Non-competitive Inhibitor that binds at alternate site other than active site Poisons Organo-phosphorous compounds Insecticides Bind to enzymes of the nervous system and kills the organism Friday, November 23, 2018

Inhibitors Friday, November 23, 2018

Enzyme Kinetics Friday, November 23, 2018

Enzyme Kinetics Way of describing properties of enzymes Mathematical Graphical expression Expression of reaction rates of enzymes A  B + C Please read Chapter 8 Section #4 Friday, November 23, 2018

Graphical Curves of Enzyme Activity Rate vs. Enzyme Ml substrate/min Rate Rate vs. pH Reveals the optimum pH Rate vs. Temperature Reveals the optimum temperature Rate vs. Substrate Shows a saturation curve Most definitive curve of enzyme activity Friday, November 23, 2018

Michaelis-Menten Enzyme Curve Michaelus and Menten proposed a simple model that accounts for most of the features of enzyme-catalyzed 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 Friday, November 23, 2018

Michaelis-Menten Equation 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 Friday, November 23, 2018

Michaelis-Menten Enzyme Curve Friday, November 23, 2018

Michaelis-Menten Equation 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 Friday, November 23, 2018

Conclusions about Michaelis-Menten Kinetics Characteristics of Km Km = substrate level at 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 Friday, November 23, 2018

Conclusions about Michaelis-Menten Kinetics 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 Friday, November 23, 2018

Conclusions about Michaelis-Menten Kinetics Order of Reaction Recall from Chemistry Will leave the details of this conclusion out. Friday, November 23, 2018

Lineweaver-Burke Plot 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 Friday, November 23, 2018

Lineweaver-Burke Equation 1/V0 = Km/Vmax[s] + 1/Vmax The intercept on the x axis -1/Km The intercept on the y axis 1/Vmax Friday, November 23, 2018

Michaelis-Menten & Lineweaver-Burke Friday, November 23, 2018

The Effect of Inhibitors Friday, November 23, 2018

Competitive Inhibition 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 Friday, November 23, 2018

Competitive Inhibition Friday, November 23, 2018

Succinate Dehydrogenase Enzyme that catalyzes the oxidation of succinate to fumarate during 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 Friday, November 23, 2018

Non-Competitive Inhibition 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 Friday, November 23, 2018

Effects of Inhibitors Friday, November 23, 2018

Lead Poisoning & Lactam Antibiotics Lead forms covalent bonds with the sulfhydryl side chains of cysteine in proteins The binding of the heavy metal shows non-competitive inhibition Drugs an behave as enzyme inhibitors Lactam antibiotics Penicillin Amoxicillin Inhibit one or more enzymes of bacteria walls Friday, November 23, 2018

Metabolic Pathways Friday, November 23, 2018

Regulation of Enzyme Activity The regulation of the reaction velocity of enzymes is essential if the organism is to coordinate its numerous metabolic pathways—The control of an organism’s metabolism. Friday, November 23, 2018

Feedback Inhibition An end product inhibits an initial pathway enzyme by altering efficiency of enzyme action Friday, November 23, 2018

Allosteric Regulation 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 Friday, November 23, 2018

Allosteric Regulation Friday, November 23, 2018