FBICH 410 1st Edition Lecture 21Outline of Last Lecture - Enzymes KineticsOutline of Current Lecture - Uncompetitive Inhibition- binds at enzyme-substrate site- not free Eo Results in decreased Vmax and Kmo Effect of uncompetitive inhibitor cant be overcome by high conc of substrate unlike competitiveo Graph- series of parallel lineso Only 1 place to get Vmax and Km—when no Inhibitor (I) presento 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 graphso The I can bind to both free E and ES complexo 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 inhibitoro Competitive= ki slope= Ki- y int doesn’t change but slope doeso 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 substrateo 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 botho Determine y int w and wo I present Ki=I/(yint w I/wo I)-1o Determine slope w and wo I present Ki=I/(slope w I/wo I)-1These 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 Mechanismso An enzyme kinetic mechanism is order of S addition and product releaseo Chemical mechanism is the pathway to convert SP including intermediateso Both are needed to understand enzyme  Kinetic can disprove chemical mechanismo 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 productsare released single displacement; uses Cleland nomenclatureo Ordered- defined order of substrate addition and product releaseo Random- rxn shows no preference- Ping pong rxn- 1 or more products are released before all S have been addedand an alternative enzyme form is produced at half rxn double replacemento 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 releaseo 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 rateo 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 catalysiso Enzyme accelerates near attack