F BICH 410 1st Edition Lecture 21 Outline of Last Lecture Enzymes Kinetics Outline of Current Lecture Uncompetitive Inhibition binds at enzyme substrate site not free E o Results in decreased Vmax and Km o Effect of uncompetitive inhibitor cant be overcome by high conc of substrate unlike competitive o Graph series of parallel lines o Only 1 place to get Vmax and Km when no Inhibitor I present o 2 ways to calc Ki Determine yint w and wo I present Determine xint w and wo I present Ki cant be negative Ki I yint w I wo I 1 Mixed and Noncompetitive 2 distinct graphs o The I can bind to both free E and ES complex o The affinity of the I to the 2 complex might be different not always the same If binding of I changes affinity for substrate Km will be changed and called mixed inhibition If only Vmax affected called non competitive inhibitor o Competitive ki slope Ki y int doesn t change but slope does o Uncompetitive ki yintercept Ki yint changes but not slope Mixed has characteristics of both Decrease in Vmax and either an increase of decrease in Km Effect of noncompetitive inhibitor can only be partially overcome by high conc of substrate o Mixed inhibition graph lines intersect If intersect above x axis Ki Ki more similar to competitive enzyme because smaller Ki value means tighter binding therefore I binds tighter to free enzyme Ki Ki when line intersects below xaxis more similar to uncompetitive because binds tighter to ES complex Mixed inhibition must calc clope of line and yint 2 ways to calculate 2Ki must calc both o Determine y int w and wo I present Ki I yint w I wo I 1 o Determine slope w and wo I present Ki I slope w I wo I 1 These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute o If Ki slope Ki yint then binds equally to E and ES therefore x intercept converges at one point on xaxis non competitive Kinetic versus Chemical Mechanisms o An enzyme kinetic mechanism is order of S addition and product release o Chemical mechanism is the pathway to convert S P including intermediates o Both are needed to understand enzyme Kinetic can disprove chemical mechanism o Bisubstrate reactions MM model was derived for single S P but most 60 of rxns are actually bisubstrate large proportion of bisubstrate rxns are transferase or oxidation reduction rxns Substrate addition and product release Sequential rxns all substrate bind to enzume before rxn occurs and products are released single displacement uses Cleland nomenclature o Ordered defined order of substrate addition and product release o Random rxn shows no preference Ping pong rxn 1 or more products are released before all S have been added and an alternative enzyme form is produced at half rxn double replacement o Modified enzyme form has covalent bond to S Initial velocity plot form of lineweaver burke plot Sequential rxn exhibits intersecting pattern of lines On these plots eventually will only get 1 line which is max saturation of S and max V Ping pong rxn shows parallel lines Product inhibition can be used to determine order of S bind and P release o Enzyme Catalytic Mechanisms Review rate enhancement e deltadeltaG RT kcat kuncat S binding via weak forces and causes Entropy reduction S oriented in optimum way increase rxn rate o Not favored thermodynamically but rather kinetically Desolvation of S gets ride of H20 that would interrupt rxn Formation of weak interaction geometric complement shape Free enzume changes upon binding want to achieve transition state optimization Achieves optimization by having enzyme most complementary to transition state Transition state analogs stable molec geometrically and electronically resemble transition state and are strong inhibitors of enzymes Rate enhancement by entropy reduction proximally and orientation effects Protein motions binding of substrates and enzyme active sites in optimum orientation lock and key vs induced kit model Protein motions facilitate catalysis o Enzyme accelerates near attack conformation