PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29On-Line References for Steady-State Enzyme KineticsDr. Peter Birch, University of Paisleyhttp://www-biol.paisley.ac.uk/kinetics/contents.htmlUniversity of Texashttp://www.cm.utexas.edu/academic/courses/Fall2001/CH369/LEC05/Lec5.htmTerre Haute Medical Collegehttp://web.indstate.edu:80/thcme/mwking/enzyme-kinetics.htmlEnzymes-- Biological CatalystsCatalyst- a substance that increases the rate of a reaction without itself being changed or consumed in the overall processTurnover- the catalyst may be reused in subsequent reactionsX* transition statereactantsproductsFree EnergyReaction ProgressG*GoFree energyof activationFree energychangeuncatalyzedreactantsproductsFree EnergyReaction ProgresscatalyzedChemical Kinetics & Equilibria:The position of equilibrium is determined by the free energy change, GoGo = -RTlnKoThe rate of a reaction depends on the free energyof activation, G*Catalysts:Speed up reactions by lowering the free energyof activation, G*Do not affect the position of equilibrium(Go unchanged)Why do we need enzymes? * A chemical reaction occurs only if the molecules possess a minimum amount of energy---Activation Energy* Chemical reactions require initial input of energy--usually in the form of increased heat* Raising the temperature increases the rate of (vibrational, translational) movement of the molecules and the chance of collision* An increase in the concentration of reactants can also increase the chances of a chemical reaction occurring* HEAT and MORE REACTANTS can increase chance of chemical reaction occurring* Biological systems cannot raise heat or concentrations at willHow do enzymes do that? * Provide alternate pathway by lowering energy of activation, stabilization of transition state--same as adding heat* Lowers activation energy, but does not change free energy required for the reaction to occur (alters the rate, but not thermodynamics)* Provide a surface for the reaction to occur, bringing reactants into close proximity to each other--functional equivalent of increasing concentrationEnzyme catalysts contain unique active sites---where the substrates bind and the reaction takes place* Lock and key model--substrate fits exactly into active site* Induced fit model--substrate causes change in enzyme's active site shape to make substrate fit Once bound, the substrate reaches the transition state and bonds are rearranged. The enzyme active site:* Places atoms in close proximity to each other* Orients substrate correctly* These two effects facilitate the breaking and reforming of bondsEnzyme-Substrate Binding SpecificityLock & KeyInduced FitClasses of enzymes * Oxidoreductases--oxidation/reduction; requires a co-factor such as NAD or FADA: + B => A + B:* Transferases--transfer of a functional groupA-B + C => A + B-C* Hydrolases--hydrolysis of functional group by waterA-B + H20 => A-H + B-OH* Lyases--elimination to form double bond; or addition to a double bondX-A-B-Y => A=B + X-Y* Isomerases--isometric interconversionsX-A-B-Y => Y-A-B-X* Ligases--ATP dependent joining of two moleculesA + B +ATP => A-B + ADP + PiEnzymes:Compilations of Databases & Online Resourceshttp://restools.sdsc.edu/biotools/biotools12.htmlFRONTIERS IN BIOSCIENCEShttp://www.bioscience.org/urllists/protdb.htmExPASy Molecular Biology Serverhttp://us.expasy.org/Enzyme Kinetics:Studies of the rate or velocity of enzyme-catalyzed reactions, and factors influencing these rates.Mathematical analyses of the relationships between substrate (or inhibitor, activator) concentrations and reactions rates yield:-- characteristic properties of enzymes or classes of enzymes-- insights into enzyme mechanisms and physiologyThe Effects of Substrate Concentration on Reaction RateS PES = substrateP = productE = enzymeUncatalyzedReaction:v[S]At a fixed concentration of enzyme, the velocity reachs a maximum, which fits the equation of a rectangular hyperbolic curve: y=(ax)/(b + x)Velocity v = d[P]/dt = -d[S]/dtMichaelis-Menton EquationFor enzyme-catalyzed reactionE + S ES E + P v = V[S] / (Km + [S]) (rectangular hyperbola)They made the following assumptions: * [E] is always much less than [S]* formation of [ES] is required to obtain [P]* therefore, at high [S], all the [E] will be saturated and the reaction cannot proceed any faster by adding more [S], this velocity is V or Vmax (“a” in general equation for hyperbolic curve)* velocity of reaction as a function of [S] is only dependent on dissociation of [ES] into [E] and [S]. The equilibrium constant, Km, is "b" in the equation for a hyperbolic curveWhen v= Vmax/2 (the velocity at 1/2 maximal velocity), then [S] = Km Km is called the Michaelis constantSteady-State Derivation of Michaelis-Menton Equation (Briggs & Haldane)timeE + S ES E + Pk1k-1k2Upon mixing of enzyme and substrate [ES] rises rapidly and reaches a steady state, where the rate of formation and breakdown of [ES] are equal, i.e. v1= k1 [E][S] and v2= k-1 [ES] + k2[ES] so that at steady state v1 = v2Free enzyme conc. [E]= [Et] -[ES]; note that [E] cannot be measured, but [Et] is known, as is [S] since initial velocities, and [P] can be measured. Now solve for the unknown [ES].v1= k1 ([Et] -[ ES])[S] = v2 = k-1[ES] + k2[ES] rearrange ([Et] -[ES])[S]/[ES] = k-1+ k2 /k1 = Km Solving for [ES] gives [ES] = [Et][S]/ Km + [S] the velocity (v) of the reaction will be proportional to the formation of [ES], so v= k2 [ES] (substitute in the value for ES in red above) to get: v=k2 [[Et][S]/ Km + [S] ] and at saturating [S], Vmax = k2[Et] (substitute Vmax for the k2 [[Et] in the above equation), then v= Vmax [S]/ Km + [S] which is the same as the Michaelis-Menton equationNote importance ofinitial velocities (vo)The Effects of Enzyme ConcentrationVmax is directly proportionalto enzyme concentrationKm is independent ofenzyme concentrationAt saturating substrate([S] >> Km):Measuring Kinetic Parameters: Graphical & Computational Methods* Lineweaver-Burk Plot* Eadie-Hofstee Plot* Hanes Plot* Direct Linear Plot* Direct Fitting of v vs. [S] Curve* Single Progress Curve* StatisticsLineweaver-Burk Plot:Rearrangement of Michaelis-Menten equation to linear form 1/v = (Km/V)(1/[S]) + 1/VPlot