Lecture 8Outline of Last Lecture1. Historical Background Catalysts as Protein Enzymes2. Enzyme Catalyzed Reactions3. Principles of Enzyme Active Site4. Equilibrium Point and Free Energy Changes are Related5. Acid-Base Catalysis6. ChymotrypsinOutline of Current Lecture1. Properties of Enzymes2. Michaelis-Menten Equation3. Lineweaver-Burk Double-Reciprocal Plot4. Enzyme Inhibition5. Pre-Steady State kineticsCurrent Lecture1. Properties of Enzymes – Catalytic PowerThree qualities of enzymes: catalytic power, reaction specificity, regulation in response to signalsCatalytic power - acceleration of chemical reaction ratesExample: ureaseWithout urease: rate constant = 3x10^-10 /secWith urease: rate constant = 3x10^4 /secCatalytic power=catalyzed rate/non-catalyzed rate= 1014V= k[S] First order reactionV=k[S1][S2] Second order reactionWhat happens first when you perform this reaction?Initially free E interacts with free S to accumulate some ES (ligand binding event)Then ES reacts chemically to EP (catalysis)Or releases free S (ligand release event)[P] initiallys = 0 so EP cannot form by product binding eventif k-1 >> k2 then ES soon accumulates to a certain concentration [ES]Back-reaction rate increases over time, so this prevents the forward reaction from reaching absolute completionHow might we measure [P]?color changes in spectrometer, further reaction with another enzymeHow can we know [E]?titrate the active sitesHow would progress curve be different if [E] were 2[E]?Rate will increase – plateau would be the same0.5[E]? less steep (slower)How would progress curve be different if [S] were 2[S]?Initial velocity and steady state approximationWhen the initial forward reaction velocity is measured (v0) the back reaction rate is negligibleV0 is the tangent to the curve at t=0Over the initial reaction interval, the rate of catalysis is ~ constant. This initial reaction phase is therefore called the steady stateAt other times, the reaction is still going forward, but the combined effects of the P  S back reaction plus depletion of S ([S]<S1) gives lower observed rates of product accumulation (v1, v2)When you change [S], you change the observed initial rate and the maximum amount of product accumulatedMaximum velocity (Vmax) – velocity at which all E is always saturated with SDirect rate plot: rates (v0) derived from many progress curvesEach progress curve for [S]n yields one rate point v0 on this direct RATE plot and the result is the curve displayedThis direct plot response diagnoses substrate saturation2. Michaelis-Menten EquationCatalytic step is slow relative to ligand binding events (ES complex formation and EP complex dissociation are faster)In that case, some catalytic step is the rate-limiting step (which implies that r.-l. step has the highest activation energy)This allows M&M to express a relationship between rate and [S]Km – Michaelis constant – inversely proportional to the affinity of the enzyme for its substrate (approximation)Concentration of substrate when v0 is equal to one-half of VmaxE is half-saturated with S when [S] = Km (km is concentrationThus 50% of active sites are filled by substrate, 50% empty[S] under physiological conditionsTurnover number (kcat) – directly proportional to the kinetic efficiency of the enzymeEquivalent to k2 under conditions of initial velocityCalled the turnover number of the enzymeNumber of substrate molecules converted into product per enzyme molecule per unit time when the enzyme is saturated with substrateKcat/Km – specificity constant ­ -- represents the catalytic efficieny of an enzyme at substrate concentration significantly below saturation3. Lineweaver-Burk Double-Reciprocal PlotLinear response: much more accurate graphical method for determining Vmax, KmExperimental considerationsThe reaction involves only one substrate, of if there are more than one substrate, the concentration of all but one kept constant [S] > [Etotal] & [Etotal] is held constantAll other variable are kept constant – such as temperature, pH, ionic strengthReactions with more than one substrateEnzyme reaction involving a ternary complexSingle displacement mechanism can be either random or orderedEnzyme reaction in which no ternary complex is formedPing-Pong or Double-Displacement Mechanism (parallel lines)4. Enzyme InhibitionInhibitor – binds enzymes or ES or enzyme-intermediate complex interferes with activity of enzymeReversible or irreversible examplesIrreversible inhibition – inhibitor binds and attaches covalentlyEnzyme is thus inactivatedTypically do not see an effect on KmExample: Serine Proteases – in chymotrypsinReversible Inhibition – 3 recognized classesCompetitive – substrate analogs (structurally similar) – bind to and overlap the normal binding siteExample: ATP and AMPPNP (non-hydrolysable analog – but looks like ATP) can donate gamma phosphateincrease in apparent Kmno effect on kcat or Vmaxinhibition coefficient = αUncompetitiveinhibitor binds to ES directly but not to EI binding results in conformational change that distorts active siteDecrease in kcat (Vmax) and cannot be reversed by higher [S] also see decrease in the apparent KmMixed (noncompetitive: pure or mixed)intercept in the left quadrantinhibitor does not look like substrateinhibitor can bind to E or ESinhibitor does NOT bind acive site on EUsually affects both Km and kcatHigher [S] cannot restore full activityRational Drug Design or SelectionSmall molecule inhibitors valuableLibraries of 10,00s – 100,000 cmpdsDesign screens to select compoundsFeatures to consider:Tight binding/reversibilityOff-target effects/toxicityPatenting possibilitiesDo all enzymes show M-M kinetics?Non-hyperbolic curves seen as evidence of cooperativity or allosteric regulationCharacteristics of multi-subunit enzymesLook for sigmoidal behaviorMay not be able to detect sigmoidal behavior at the conditions of experimentsHow does allostery effect kinetics?5. Pre-Steady State kineticsRefers to brief initial lag period while [ES] builds upShort interval (usually microseconds to milliseconds)Difficult to address experimentally – this interval not usually revealed by any of the standard plotsBIOSC 1000 Lecture 8Outline of Last Lecture1. Historical Background Catalysts as Protein Enzymes2. Enzyme Catalyzed Reactions3. Principles of Enzyme Active Site4. Equilibrium Point and Free Energy Changes are Related5. Acid-Base Catalysis6. Chymotrypsin Outline of