Chapter 8 BISC 330 Midterm 2 Study Guide Enzymes are powerful catalysts with high substrate specificity o Increase rate of biochemical reactions o Do not alter equilibrium of a reaction or affect energetics Enzymes often require co factors coenzymes or metals Change in free energy can be used to estimate if reaction occurs and evaluate the energy cost of the reaction Enzymes o Generally proteins sometimes RNA o Act as catalysts for biochemical reactions Form complexes by binding a substrate at an active site Highly specific binding enzyme not chemically altered by reaction do not change the Keq of a reaction o Increase rate of reaction by lowering activation energy Reaction rate enhancement enzyme catalyzed rate constant uncatalyzed rate constant o kcat kun Catalytic substrate specificity of enzymes o Enzymes extremely specific Substrate they recognize Reactions they catalyze o Ie Thrombin proteolytic enzyme involved in blood clotting cascade Hydrolyzes C N between Arg and Gly Trypsin enzyme involved in digestive system less specific than Cofactors small molecules that enzymes need to execute biochemical reactions thrombin Cuts after Lys or Arg o Either coenzymes or metals o Apoenzyme cofactor to holoenzyme Enzyme energetics o Energy difference between reactant and products determine whether reaction takes place and the degree to which enzyme accelerates reaction o Difference in free energy determine if reaction is spontaneous or not o Note does not tell anything about rate of a reaction o Determine free energy change through Standard free energy change concentration of reactants and products T Enzyme kinetics alter rate of biochemical reaction not equilibrium o Accelerate the time it takes to reach equilibrium no effect on equilibirum o Vf kr S Vr kR P Transition state particular chemical configuration of the substrate as it evolves towards becoming the product during reaction o Very short lived chemical state o Usually highest free energy peak of free energy diagram o Delta G catalyzed Delta G transition state delta G substrate Decrease Delta G catalyzed substrate can more easily reach the transition state to become product o Relationship between G and rate constant k o k is inversely proportional to G and exponentially dependent lower the G the higher the k small changes in G leads to very large change in k Ways to increase the rate of reactions o Increase the concentrate of substrate o Increase rate constant by increasing the temperature or decreasing the activation o Enzyme increase rate of reaction by decreasing activation energy Enzymes decrease G by the transition state o Binding the substrate and providing an environment that favors the formation of o Kinetic evidence for enzyme substrate interaction ES complex o Catalytic rate increase with increased substrate concentration until a plateau is energy reached o Must be an interaction between E and S that leads to saturation of the enzyme o Physical evidence X ray crystallography Active catalytic site in enzyme o 3D cleft pocket formed by far apart amino acid residues steric hindrance Small area in enzyme often non polar environment o Binding involves multiple weak interactions between E and S H bonds electrostatic hydrophobic Van der Waals Allows strong binding yet flexibility to promote the formation of transition state o Provides high specificity for substrate shape of active catalytic pocket is flexible and can change conformation after binding of the substrate to become sterically and chemically complementary to the substrate induced fit Breathing key for high specific binding of substrate and strong binding of the transition state and for release of product Binding energy between E and S o Binding by multiple weak interactions provides free energy Some free energy is released upon formation of a bond state Used to help form active site induced fit Used to lower free energy of the transition state Implies that enzyme binds the transition state very strongly yet very shortly Using multiple steps for biochemical reaction is strategy employed to increase the rate of a biochemical reaction and to ensure high substrate specificity o In free energy diagram each step has its own transition state o Enzymes speed up biochemical reactions by Specific substrate recognition Multiple reactive steps at catalytic site Strong binding to transition state Efficient release of product Chemical Reaction Rates o V k S B Enzymatic reaction rate o Good way to study enzymatic rate is to follow appearance of P over time for differing substrate concentrations o V slows down as S is converted to P o Reverse reaction becomes significant as P accumulates producing an equilibrium Michaelis Menten Curve o At low S Vo proportional to S o At mid ranged Vo increase less than S o At high S Vo independent of S o Describe the reaction with parameters that we know or can measure S total E Km Vmax o Steady state assumption Ves formed Ves broken Reciprocal graph accurate determination of Km and Vmax o X int 1 Km o Y int 1 Vmax o Slope Km Vmax Km the maximum rate Vmax kcat Km o Substrate concentration at which the kinetic rate of an enzymatic reaction is half Measure of the concentration of substrate required to achieve significant catalysis with a given enzyme High Km weak binding Low Km strong binding o Directly related to the turnover rate of an enzymatic reaction Number of substrate molecules transformed into product by one molecule of enzyme per unit time when enzyme is fully saturated o Allows us to compare the catalytic efficiency Of a single enzyme for different substrates Of different enzymes o Max catalytic rate is intrinsically limited by probability of an enzyme to encounter a substrate limited by diffusion o 108 109 s 1M 1 are close to kinetic perfection catalysis nearly as fast as diffusion Enzymatic inhibitions o Reversible and irreversible o Inhibitors small compounds that affect the activity of enzymes o Competitive increase Km but no effect on Vmax o Uncompetitive decrease both Km and Vmax o Noncompetitive Decrease Vmax but no effect on Km Competitive inhibition o Enzyme binds S or I can be freed from I by increasing S o DHFR involved in nucleotide biosynthesis very similar to Methotrexate Cancer drug used to kill rapidly dividing cells Uncompetitive inhibition o I binds to ES complex only possible only after S binding o ESI complex cannot make P o High S does not overcome inhibition Noncompetitive inhibition o E binds both I and S at