Enzyme Kinetics and Inhibition

Enzyme Kinetics II: Kinetics and Inhibition

  • Define key terms related to enzymology and the characteristics of enzymes.

  • Describe enzymatic assays and analyze enzyme kinetics data.

  • Understand enzyme function in cellular processes.

Enzymes – Reaction Order

  • In a simple reaction, a substrate AA is reduced as a product PP is produced.

  • Reaction velocity can be defined as:

    • ν=−Δ[A]Δtν = -\frac{Δ[A]}{Δt} or ν=Δ[P]Δtν = \frac{Δ[P]}{Δt}

  • First-order rate equation: ν=k[A]ν = k[A], where kk is the rate constant.

    • The rate is directly proportional to the concentration of A ([A][A]).

    • The velocity is in Ms-1, and the reaction constant is in s-1.

    • This is a unimolecular reaction: A→PA → P

  • kk does not change.

  • Velocity reduces as AA is used up, and BB becomes the substrate for the reverse reaction.

  • The reaction reaches equilibrium if the rates for the forward and reverse reactions are equal, and the overall rate is zero.

Enzymes – Reaction Order (Bimolecular)

  • If A+B→PA + B → P, the reaction velocity can be defined as:

    • ν=−Δ[A]Δtν = -\frac{Δ[A]}{Δt} or ν=−Δ[B]Δtν = -\frac{Δ[B]}{Δt}

  • Second-order rate equation: ν=k[A][B]ν = k[A][B], where kk is the rate constant.

    • The rate is first order in [A][A] and first order in [B][B].

    • The velocity is in Ms-1, and the reaction constant is in M-1 s-1.

    • This is a bimolecular reaction: A+B→PA + B → P

Enzymes – Kinetics Problem

  • In terms of enzyme kinetics, we can think of a reaction as: S+E→P+ES + E → P + E

  • If we apply the law of mass action, the rate of reaction would linearly increase as [S][S] increases.

  • As [S][S] increases, νν increases.

  • The reaction cannot proceed this way due to enzyme saturation.

  • The leveling of the curve is due to the reverse reaction. ENZYME CONCENTRATION IS RATE LIMITING.

Enzymes – Measuring Activity

  • For a saturated enzymatic reaction, we only plot the initial rate, i.e., the reverse reaction can be ignored.

  • This is the slope of the graph.

  • Once the rate is calculated at multiple substrate concentrations, the rate can be plotted against substrate concentration.

  • VmaxV_{max} is the maximal velocity of a reaction.

  • MKM,theMichaelisconstant,isameasureofanenzyme’sefficiencyandisthesubstrateconcentrationthathasareactionratehalfthe, the Michaelis constant, is a measure of an enzyme’s efficiency and is the substrate concentration that has a reaction rate half the V{max}.</p></li></ul><h4id="9fa96c89−e2f3−47fb−b868−05ca25afa3ec"data−toc−id="9fa96c89−e2f3−47fb−b868−05ca25afa3ec"collapsed="false"seolevelmigrated="true">Enzymes–Michaelis−Menten</h4><ul><li><p>Whenthesubstrateconcentrationisoftenmuchhigherthanenzymeconcentrations,i.e.,thesubstrateisinexcess,wecandescribethereactionintwosteps:</p><ul><li><p>.</p></li></ul><h4 id="9fa96c89-e2f3-47fb-b868-05ca25afa3ec" data-toc-id="9fa96c89-e2f3-47fb-b868-05ca25afa3ec" collapsed="false" seolevelmigrated="true">Enzymes – Michaelis-Menten</h4><ul><li><p>When the substrate concentration is often much higher than enzyme concentrations, i.e., the substrate is in excess, we can describe the reaction in two steps:</p><ul><li><p>EandandSformtheintermediateform the intermediateES((k_1)–thisisreversible.</p></li><li><p>) – this is reversible.</p></li><li><p>ESisthemostpopulousform.</p></li><li><p>is the most populous form.</p></li><li><p>ESreactsandproducesreacts and producesP(andreleases(and releasesE)inanon−reversiblereaction() in a non-reversible reaction (k_2).</p></li></ul></li><li><p>).</p></li></ul></li><li><p>E + S \rightleftharpoons ES \rightarrow E + Pwithrateconstantswith rate constantsk1,,k{-1},and, andk_2.</p></li></ul><h4id="a07c3837−2855−4577−8155−a55121a33ecb"data−toc−id="a07c3837−2855−4577−8155−a55121a33ecb"collapsed="false"seolevelmigrated="true">Enzymes–Michaelis−MentenEquation</h4><ul><li><p>Intermsofenzymekinetics,theMichaelis–Mentenequationdescribestherateoftheenzymaticreaction:</p><ul><li><p>Theformationof.</p></li></ul><h4 id="a07c3837-2855-4577-8155-a55121a33ecb" data-toc-id="a07c3837-2855-4577-8155-a55121a33ecb" collapsed="false" seolevelmigrated="true">Enzymes – Michaelis-Menten Equation</h4><ul><li><p>In terms of enzyme kinetics, the Michaelis–Menten equation describes the rate of the enzymatic reaction:</p><ul><li><p>The formation ofPisafirst−orderreaction.</p></li><li><p>Therateofproductformationcanbeexpressedas:is a first-order reaction.</p></li><li><p>The rate of product formation can be expressed as:ν = \frac{Δ[P]}{Δt} = k2[ES].∗TherateofESproductionisthedifferencebetweentherateof. *The rate of ES production is the difference between the rate ofk1,andof, and ofk{-1}andandk2::\frac{Δ[ES]}{Δt} = k1[E][S] - k{-1}[ES] - k_2[ES].</p></li></ul></li></ul><h4id="ace70177−529d−4f22−9b7d−fd7c86363bc4"data−toc−id="ace70177−529d−4f22−9b7d−fd7c86363bc4"collapsed="false"seolevelmigrated="true">Enzymes–Michaelis−MentenAssumptions</h4><ul><li><p>TheMichaelis–Mentenequationrequirestwoassumptions:</p><ul><li><p><strong>Equilibrium</strong>:.</p></li></ul></li></ul><h4 id="ace70177-529d-4f22-9b7d-fd7c86363bc4" data-toc-id="ace70177-529d-4f22-9b7d-fd7c86363bc4" collapsed="false" seolevelmigrated="true">Enzymes – Michaelis-Menten Assumptions</h4><ul><li><p>The Michaelis–Menten equation requires two assumptions:</p><ul><li><p><strong>Equilibrium</strong>:k{-1} >> k2,sothefirststepofthereactionreachesequilibrium,where, so the first step of the reaction reaches equilibrium, whereKSisthedissociationconstantofthefirststepinthereaction:is the dissociation constant of the first step in the reaction:KS = \frac{k{-1}}{k1} = \frac{[E][S]}{[ES]}.</p></li><li><p><strong>Steadystate</strong>:therateof.</p></li><li><p><strong>Steady state</strong>: the rate ofESformation=therateofformation = the rate ofESdissociation:dissociation:\frac{Δ[ES]}{Δt} = 0.</p></li></ul></li></ul><h4id="64ba1dbe−0f17−4a0b−84a0−7bb5c0deac7f"data−toc−id="64ba1dbe−0f17−4a0b−84a0−7bb5c0deac7f"collapsed="false"seolevelmigrated="true">Enzymes–KMMichaelisConstant</h4><ul><li><p>.</p></li></ul></li></ul><h4 id="64ba1dbe-0f17-4a0b-84a0-7bb5c0deac7f" data-toc-id="64ba1dbe-0f17-4a0b-84a0-7bb5c0deac7f" collapsed="false" seolevelmigrated="true">Enzymes – KM Michaelis Constant</h4><ul><li><p>[ET] = [E] + [ES]andand[E] = [ET] - [ES].</p></li><li><p>Assumingsteady−stateconditions:.</p></li><li><p>Assuming steady-state conditions:k1 [E][S] = k{-1}[ES] + k_2[ES].</p></li><li><p>Consideringtheabove:.</p></li><li><p>Considering the above:([ET] - [ES])[S] = \frac{k{-1} + k2}{k1} [ES].</p></li></ul><h4id="19ac7328−2065−4a84−a43e−ad1cb6218779"data−toc−id="19ac7328−2065−4a84−a43e−ad1cb6218779"collapsed="false"seolevelmigrated="true">Enzymes–KMMichaelisConstant(cont.)</h4><ul><li><p>.</p></li></ul><h4 id="19ac7328-2065-4a84-a43e-ad1cb6218779" data-toc-id="19ac7328-2065-4a84-a43e-ad1cb6218779" collapsed="false" seolevelmigrated="true">Enzymes – KM Michaelis Constant (cont.)</h4><ul><li><p>K_Mistheconcentrationofsubstrateatwhichtherateishalfthemaximalrate.</p></li><li><p>is the concentration of substrate at which the rate is half the maximal rate.</p></li><li><p>KM = \frac{V{max}}{2} = \frac{k{-1} + k2}{k_1}.</p></li><li><p>.</p></li><li><p>KMisrelatedtotheaffinityofanenzymeforasubstrate;thehighertheis related to the affinity of an enzyme for a substrate; the higher theKM,thelowertheaffinity.</p></li><li><p>, the lower the affinity.</p></li><li><p>K_Misnotaffectedbytheis not affected by the[E].</p></li><li><p>Itdependsbothontheenzymeandthesubstrate.</p></li><li><p>Reactionconditions,suchastemperatureandpH,canalsoaffect.</p></li><li><p>It depends both on the enzyme and the substrate.</p></li><li><p>Reaction conditions, such as temperature and pH, can also affectK_M.</p></li></ul><h4id="7214a0f8−633c−4ccb−ab99−76087f1803ca"data−toc−id="7214a0f8−633c−4ccb−ab99−76087f1803ca"collapsed="false"seolevelmigrated="true">Enzymes–Michaelis−MentenEquation(cont.)</h4><ul><li><p>TheMichaelis–Mentenequationisusedtocalculatethereactionrateofproductformation:.</p></li></ul><h4 id="7214a0f8-633c-4ccb-ab99-76087f1803ca" data-toc-id="7214a0f8-633c-4ccb-ab99-76087f1803ca" collapsed="false" seolevelmigrated="true">Enzymes – Michaelis-Menten Equation (cont.)</h4><ul><li><p>The Michaelis–Menten equation is used to calculate the reaction rate of product formation:ν = \frac{Δ[P]}{Δt} = k_2[ES].</p></li><li><p>Considering.</p></li><li><p>ConsideringKM = \frac{([ET] - [ES])[S]}{[ES]} = \frac{k{-1} + k2}{k_1}.</p></li><li><p>Wecanrearrangeandsolvefor.</p></li><li><p>We can rearrange and solve for[ES],giving:, giving:[ES] = \frac{[ET][S]}{KM + [S]}.</p></li></ul><h4id="c2a843c8−57f8−481c−be45−a11e907eb7d2"data−toc−id="c2a843c8−57f8−481c−be45−a11e907eb7d2"collapsed="false"seolevelmigrated="true">Enzymes–Michaelis−MentenEquation(InitialVelocity)</h4><ul><li><p>Fortheinitialvelocityofareaction,whichwemeasureexperimentally:</p><ul><li><p>Theinitialratedependson.</p></li></ul><h4 id="c2a843c8-57f8-481c-be45-a11e907eb7d2" data-toc-id="c2a843c8-57f8-481c-be45-a11e907eb7d2" collapsed="false" seolevelmigrated="true">Enzymes – Michaelis-Menten Equation (Initial Velocity)</h4><ul><li><p>For the initial velocity of a reaction, which we measure experimentally:</p><ul><li><p>The initial rate depends on[E]andand[S].</p></li><li><p>TheinitialratedoesNOTincreaseindefinitelywith.</p></li><li><p>The initial rate does NOT increase indefinitely with[S]buttailsofftoamaximum.</p></li></ul></li><li><p>Formostenzymaticreactions,themaximumratebut tails off to a maximum.</p></li></ul></li><li><p>For most enzymatic reactions, the maximum rateV{max}(when(when[S] >> KM)istherefore:) is therefore:V{max} = k2[E_T].</p></li><li><p>Therefore,theMichaelis−Mentenequationis:.</p></li><li><p>Therefore, the Michaelis-Menten equation is:νo = \frac{Δ[P]}{Δt} = k2[ES] = \frac{k2[ET][S]}{KM + [S]} = \frac{V{max}[S]}{K_M + [S]}.</p></li></ul><h4id="8f8cfc38−5cba−4160−ad96−a16a33852d4b"data−toc−id="8f8cfc38−5cba−4160−ad96−a16a33852d4b"collapsed="false"seolevelmigrated="true">Enzymes–KMMichaelisConstant(DissociationofESComplex)</h4><ul><li><p>.</p></li></ul><h4 id="8f8cfc38-5cba-4160-ad96-a16a33852d4b" data-toc-id="8f8cfc38-5cba-4160-ad96-a16a33852d4b" collapsed="false" seolevelmigrated="true">Enzymes – KM Michaelis Constant (Dissociation of ES Complex)</h4><ul><li><p>K_Misalsothedissociationoftheis also the dissociation of theEScomplex.</p></li><li><p>Whencomplex.</p></li><li><p>Whenk2isnegligible(is negligible ([P]negligible):negligible):KM = \frac{k{-1} + k2}{k1} ≈ \frac{k{-1}}{k1} = KS.</p></li></ul><h4id="fb3c2438−cde8−4f1c−8131−06a3503c8af3"data−toc−id="fb3c2438−cde8−4f1c−8131−06a3503c8af3"collapsed="false"seolevelmigrated="true">Enzymes–CatalyticEfficiency</h4><ul><li><p>.</p></li></ul><h4 id="fb3c2438-cde8-4f1c-8131-06a3503c8af3" data-toc-id="fb3c2438-cde8-4f1c-8131-06a3503c8af3" collapsed="false" seolevelmigrated="true">Enzymes – Catalytic Efficiency</h4><ul><li><p>KMcanalsobeusedtocalculatethecatalyticefficiency,calledthespecificityconstant,can also be used to calculate the catalytic efficiency, called the specificity constant,k{cat}/K_M.</p><ul><li><p>Thenumberofcatalyzedreactionsperactivesiteperunittime.</p></li></ul></li><li><p>Rearranging,.</p><ul><li><p>The number of catalyzed reactions per active site per unit time.</p></li></ul></li><li><p>Rearranging,k{cat}isequivalenttois equivalent tok2insimplereactions:in simple reactions:k{cat} = \frac{V{max}}{[E_T]}.</p></li><li><p>.</p></li><li><p>k_{cat}doesnotchange.</p></li><li><p>Catalyticefficiencyislimitedbydiffusion.</p></li><li><p>Thus,akineticallyperfectenzymehasanupperlimitofdoes not change.</p></li><li><p>Catalytic efficiency is limited by diffusion.</p></li><li><p>Thus, a kinetically perfect enzyme has an upper limit ofk{cat}/KM(108or109).</p></li></ul><h4id="00f3c451−1da9−4bff−aa03−6ddef1c20771"data−toc−id="00f3c451−1da9−4bff−aa03−6ddef1c20771"collapsed="false"seolevelmigrated="true">Enzymes–Lineweaver−BurkPlot</h4><ul><li><p>TheLineweaver−Burkplotgivesacommongraphtypethatallowseasycalculationofenzymekineticparameters((108 or 109).</p></li></ul><h4 id="00f3c451-1da9-4bff-aa03-6ddef1c20771" data-toc-id="00f3c451-1da9-4bff-aa03-6ddef1c20771" collapsed="false" seolevelmigrated="true">Enzymes – Lineweaver-Burk Plot</h4><ul><li><p>The Lineweaver-Burk plot gives a common graph type that allows easy calculation of enzyme kinetic parameters (y = mx + c).</p></li><li><p>Thex−interceptisequivalentto).</p></li><li><p>The x-intercept is equivalent to\frac{-1}{K_M}.</p></li><li><p>They−interceptisequivalentto.</p></li><li><p>The y-intercept is equivalent to\frac{1}{V_{max}}.</p></li><li><p>Thegradientisequivalentto.</p></li><li><p>The gradient is equivalent to\frac{KM}{V{max}}.</p></li><li><p>Disadvantage:small.</p></li><li><p>Disadvantage: small[S]valuesdominatetheplot,whichmayresultinlessaccuratemeasurements.</p></li></ul><h4id="4001682a−df39−4616−94cf−275954bffccf"data−toc−id="4001682a−df39−4616−94cf−275954bffccf"collapsed="false"seolevelmigrated="true">Enzymes–Inhibition</h4><ul><li><p>Enzymeinhibitorsareanimportantclassofdrugsforarangeofdiseases.</p></li><li><p>Severalclassesexistthatfunctionindifferentways:</p><ul><li><p>Competitiveinhibitors</p></li><li><p>Non−competitiveinhibitors</p></li><li><p>Uncompetitiveinhibitors</p></li></ul></li><li><p>Eachinhibitortypeaffectsthereactionkineticsdifferently.</p></li><li><p>Inhibitorscanbereversible,irreversible,orcovalent.</p></li></ul><h4id="e6eeda45−63da−4fa7−9745−d65bda67b96a"data−toc−id="e6eeda45−63da−4fa7−9745−d65bda67b96a"collapsed="false"seolevelmigrated="true">Enzymes–CompetitiveInhibition</h4><ul><li><p>Competitiveinhibitioninvolvesinhibitorbindingatanenzyme’sactivesite.</p></li><li><p>Thispreventsthebindingofthesubstrate.</p></li><li><p>Increasingvalues dominate the plot, which may result in less accurate measurements.</p></li></ul><h4 id="4001682a-df39-4616-94cf-275954bffccf" data-toc-id="4001682a-df39-4616-94cf-275954bffccf" collapsed="false" seolevelmigrated="true">Enzymes – Inhibition</h4><ul><li><p>Enzyme inhibitors are an important class of drugs for a range of diseases.</p></li><li><p>Several classes exist that function in different ways:</p><ul><li><p>Competitive inhibitors</p></li><li><p>Non-competitive inhibitors</p></li><li><p>Uncompetitive inhibitors</p></li></ul></li><li><p>Each inhibitor type affects the reaction kinetics differently.</p></li><li><p>Inhibitors can be reversible, irreversible, or covalent.</p></li></ul><h4 id="e6eeda45-63da-4fa7-9745-d65bda67b96a" data-toc-id="e6eeda45-63da-4fa7-9745-d65bda67b96a" collapsed="false" seolevelmigrated="true">Enzymes – Competitive Inhibition</h4><ul><li><p>Competitive inhibition involves inhibitor binding at an enzyme’s active site.</p></li><li><p>This prevents the binding of the substrate.</p></li><li><p>Increasing[S]canovercomethiskindofinhibition.</p></li><li><p>Kinetically,can overcome this kind of inhibition.</p></li><li><p>Kinetically,KMincreases,andincreases, andV{max}doesnotchange.</p></li><li><p>does not change.</p></li><li><p>Kiisthedissociationconstantforinhibition:is the dissociation constant for inhibition:Ki = \frac{[E][I]}{[EI]}.</p></li></ul><h4id="ec3739b9−dc6d−4eaa−9b7a−38eb60f348a5"data−toc−id="ec3739b9−dc6d−4eaa−9b7a−38eb60f348a5"collapsed="false"seolevelmigrated="true">Enzymes–CompetitiveInhibition(Lineweaver−Burk)</h4><ul><li><p>Lineweaver−Burkplotscanbeusedtocalculate.</p></li></ul><h4 id="ec3739b9-dc6d-4eaa-9b7a-38eb60f348a5" data-toc-id="ec3739b9-dc6d-4eaa-9b7a-38eb60f348a5" collapsed="false" seolevelmigrated="true">Enzymes – Competitive Inhibition (Lineweaver-Burk)</h4><ul><li><p>Lineweaver-Burk plots can be used to calculateK_i.</p></li><li><p>Here,thegradientisequalto.</p></li><li><p>Here, the gradient is equal to\frac{αKM}{V{max}}.</p></li><li><p>.</p></li><li><p>Kicanbecalculatedbytheapparentcan be calculated by the apparentKMusingtheequation:using the equation:K{M}^{app} = αKM.</p></li><li><p>.</p></li><li><p>αiscalculatedby:is calculated by:α = 1 + \frac{[I]}{K_i}.</p></li></ul><h4id="0f09a4df−5c1e−4ebb−9e3e−78f289cb98a6"data−toc−id="0f09a4df−5c1e−4ebb−9e3e−78f289cb98a6"collapsed="false"seolevelmigrated="true">Enzymes–UncompetitiveInhibition</h4><ul><li><p>Uncompetitiveinhibitioninvolvestheinhibitorbindingtotheenzyme−substratecomplex.</p></li><li><p>Theydonotbindtofreeenzyme.</p></li><li><p>Increasing.</p></li></ul><h4 id="0f09a4df-5c1e-4ebb-9e3e-78f289cb98a6" data-toc-id="0f09a4df-5c1e-4ebb-9e3e-78f289cb98a6" collapsed="false" seolevelmigrated="true">Enzymes – Uncompetitive Inhibition</h4><ul><li><p>Uncompetitive inhibition involves the inhibitor binding to the enzyme-substrate complex.</p></li><li><p>They do not bind to free enzyme.</p></li><li><p>Increasing[S]cannotovercomethiskindofinhibition.</p></li><li><p>Kinetically,bothcannot overcome this kind of inhibition.</p></li><li><p>Kinetically, bothKMandandV{max}decrease.</p></li><li><p>decrease.</p></li><li><p>K'iisthedissociationconstantforinhibition:is the dissociation constant for inhibition:K'i = \frac{[ES][I]}{[ESI]}.</p></li></ul><h4id="ea4f3dc8−a806−4714−a5b5−c587754142ea"data−toc−id="ea4f3dc8−a806−4714−a5b5−c587754142ea"collapsed="false"seolevelmigrated="true">Enzymes–UncompetitiveInhibition(Lineweaver−Burk)</h4><ul><li><p>Lineweaver−Burkplotscanbeusedtocalculate.</p></li></ul><h4 id="ea4f3dc8-a806-4714-a5b5-c587754142ea" data-toc-id="ea4f3dc8-a806-4714-a5b5-c587754142ea" collapsed="false" seolevelmigrated="true">Enzymes – Uncompetitive Inhibition (Lineweaver-Burk)</h4><ul><li><p>Lineweaver-Burk plots can be used to calculateK'_i.</p></li><li><p>Here,thegradientisequalto.</p></li><li><p>Here, the gradient is equal to\frac{KM}{V{max}}.</p></li><li><p>.</p></li><li><p>Kicanbecalculatedbytheapparentcan be calculated by the apparentKMusingtheequation:using the equation:K{M}^{app} = \frac{KM}{α'}.</p></li><li><p>.</p></li><li><p>α'iscalculatedby:is calculated by:α' = 1 + \frac{[I]}{K'_i}.</p></li></ul><h4id="9e74d047−efaf−440e−a5d1−6dae02400ab4"data−toc−id="9e74d047−efaf−440e−a5d1−6dae02400ab4"collapsed="false"seolevelmigrated="true">Enzymes–MixedorNon−competitiveInhibition</h4><ul><li><p>Mixedinhibitionaffectsbothsubstratebindingandcatalyticactivity.</p></li><li><p>Bothenzymeandenzyme−substratecomplexcanbebound.</p></li><li><p>Increasing.</p></li></ul><h4 id="9e74d047-efaf-440e-a5d1-6dae02400ab4" data-toc-id="9e74d047-efaf-440e-a5d1-6dae02400ab4" collapsed="false" seolevelmigrated="true">Enzymes – Mixed or Non-competitive Inhibition</h4><ul><li><p>Mixed inhibition affects both substrate binding and catalytic activity.</p></li><li><p>Both enzyme and enzyme-substrate complex can be bound.</p></li><li><p>Increasing[S]cannotovercomethiskindofinhibition.</p></li><li><p>Kinetically,cannot overcome this kind of inhibition.</p></li><li><p>Kinetically,KMdoesnotchange,anddoes not change, andV{max}decreases.</p></li><li><p>decreases.</p></li><li><p>KiandandK'iarethedissociationconstantsforinhibition:</p><ul><li><p>are the dissociation constants for inhibition:</p><ul><li><p>K'_i = \frac{[ES][I]}{[ESI]}</p></li><li><p></p></li><li><p>K_i = \frac{[E][I]}{[EI]}</p></li></ul></li></ul><h4id="00f8a812−2380−4313−8c22−99b9fe9bd8c5"data−toc−id="00f8a812−2380−4313−8c22−99b9fe9bd8c5"collapsed="false"seolevelmigrated="true">Enzymes–MixedorNon−competitiveInhibition(Lineweaver−Burk)</h4><ul><li><p>Lineweaver−Burkplotscanbeusedtocalculate</p></li></ul></li></ul><h4 id="00f8a812-2380-4313-8c22-99b9fe9bd8c5" data-toc-id="00f8a812-2380-4313-8c22-99b9fe9bd8c5" collapsed="false" seolevelmigrated="true">Enzymes – Mixed or Non-competitive Inhibition (Lineweaver-Burk)</h4><ul><li><p>Lineweaver-Burk plots can be used to calculateK_i.</p></li><li><p>Here,thegradientisequalto.</p></li><li><p>Here, the gradient is equal to\frac{αKM}{V{max}}.</p></li><li><p>.</p></li><li><p>KiandandK'icanbecalculatedbytheapparentcan be calculated by the apparentK_Musingtheequations:</p><ul><li><p>using the equations:</p><ul><li><p>α' = 1 + \frac{[I]}{K'_i}</p></li><li><p></p></li><li><p>α = 1 + \frac{[I]}{K_i}</p></li></ul></li></ul><h4id="193b0d00−72bc−449d−811b−d2cc6915da23"data−toc−id="193b0d00−72bc−449d−811b−d2cc6915da23"collapsed="false"seolevelmigrated="true">Enzymes–InhibitionTypes</h4><ul><li><p><strong>Competitive</strong>:</p></li></ul></li></ul><h4 id="193b0d00-72bc-449d-811b-d2cc6915da23" data-toc-id="193b0d00-72bc-449d-811b-d2cc6915da23" collapsed="false" seolevelmigrated="true">Enzymes – Inhibition Types</h4><ul><li><p><strong>Competitive</strong>:V{max}unchanged,unchanged,KMincreased</p></li><li><p><strong>Non−competitive</strong>:increased</p></li><li><p><strong>Non-competitive</strong>:V{max}decreased,decreased,KMunchanged</p></li><li><p><strong>Uncompetitive</strong>:Bothunchanged</p></li><li><p><strong>Uncompetitive</strong>: BothV{max} & KM$$ decreased

  • Each of the inhibitions can be differentiated via Direct Plots or Double Reciprocal Plots as defined in the transcript

  • Define key equations related to enzyme kinetics, including under conditions of inhibition.

  • Describe mechanisms of enzyme inhibition.

  • Analyze enzyme activity in different biological pathways and processes.

  • Complete kinetic equations and work through examples of enzyme calculations.