BIOC 460 Summer 2010 Enzymes Inhibition Naproxen Aleve an NSAID Nonsteroidal Anti Inflammatory Drug competitive inhibitor of PGH2 synthase COX cyclooxygenase first enzyme in biosynthetic pathway of prostaglandins local hormones involved in inflammation Reading Berg Tymoczko Stryer 6th ed Chapter 8 pp 225 236 Problems pp 238 239 chapter 8 1 2 4a b 5a b 7 10 Jmol structure cyclooxygenase non steroidal anti inflammatory drugs http www biochem arizona edu classes bioc462 462a jmol cox12 cox121 htm Key Concepts Km and Vmax can be determined from double reciprocal plots 1 Vo vs 1 S Enzyme inhibitors compounds that reduce velocity of enzymecatalyzed reactions 2 types reversible and irreversible reversible inhibitors competitive inhibitor I increases Km app but has no effect on Vmax there are also two other types of reversible binding inhibitors uncompetitive and non competitive inhibitors however we will not discuss these inhibitors in this course Irreversible inhibitors cause irreversible generally covalent modification of the enzyme inactivating itit Enzymes Inhibition 1 BIOC 460 Summer 2010 Key Concepts continued Both reversible and irreversible inhibitors very helpful for providing information about shape of active site and types of amino acid side chains there working out enzyme mechanisms providing idi iinfo f about b t control t l off metabolic t b li pathways th design of drugs Graphical Determination of Km and Vmax Enzyme kinetics Vo as a function of S to obtain Vmax and Km computer fit of data using Michaelis Menten eqn by hand can t extrapolate on hyperbolic plot to get accurate Vmax and Km values simple solution without a computer linear transformation aka double reciprocal plot Lineweaver Burk Plot Take reciprocal of both sides of M M Equation and rearrange to get Lineweaver Burk equation Enzymes Inhibition 2 BIOC 460 Summer 2010 Lineweaver Burk Plot double reciprocal plot Equation of a straight line y mx b x intercept 1 Km y intercept 1 Vmax slope Km Vmax If x intercept 1 Km 2 x 104 M 1 What Wh t is i K m I Km 1 2 x 104 M 1 0 5 x 10 4 M or 5 x 10 5 M Note POSITIVE value 2 I 1 I 1 Can Km ever have a negative value Enzyme Inhibitors reversible rapid binding release from enzyme in an equilibrium or irreversible very tightly bound to enzyme either covalently or noncovalently but effectively don t come off Reversible inhibitors type of inhibition diagnosed by effect of inhibitor on Km and or Vmax effects diagnosis obvious on double reciprocal plot basis for drugs actions research tools in figuring out enzyme chemical catalytic mechanisms Classes competitive what you will be held responsible for uncompetitive not responsible for noncompetitive not responsible for Enzymes Inhibition 3 BIOC 460 Summer 2010 Reversible Enzyme Inhibitors continued ES Complex Berg et al Fig 8 15 Prevents S from binding so increases Km no effect on Vmax Pure Decreases Km and reduces Vmax by same factor so slope of 1 Vo vs 1 S doesn t change binds only to ES complex Has no effect on Km no effect on S binding reduces Vmax reduces kcat Competitive Inhibition Enzyme can bind either substrate or inhibitor but not both Inhibitor binds in same site as S Enzyme active site can be free E or have S bound ES or have I bound EI There s no ESI complex Competitive inhibitor increases apparent Km Km app Doesn t affect Vmax High S overcomes effect of I Ki inhibition constant a Kd for EI complex E I EI Km Km and Ki are diss n constants for ES and EI What does Ki Km signify If Ki Km 1 1 or 1 what does this say about competing equilibria Berg et al Fig 8 17 Enzymes Inhibition 4 BIOC 460 Summer 2010 Competitive Inhibition continued Competitive inhibitor increases apparent Km but doesn t affect Vmax High S overcomes effect of I Km apparently higher with inhibitor than without inhibitor Definition of Km Vmax not changed Km app Berg et al Fig 8 17 Berg et al Fig 8 20 1 Km 1 Km app How and Why does Km Apparently Change Logical Answer Because of equilibrium between S and I for free E it takes a higher S to reach Vmax 2 So Km app takes into account both I and Ki Mathematical answer Km app Km 1 I Ki Ki is a dissociation constant for Inhibitor The tighter the inhibitor binding the smaller the Ki How would Km app change as Ki decreases from 10 uM to 1 uM to 0 1 uM fixed I How would Km app change as I increases fixed Ki Note Km does not actually change Km reflects interaction of S and E only Km app reflects the effect of I and its Ki on what you determine for the apparent Km Enzymes Inhibition 5 BIOC 460 Summer 2010 Effect of Ki on Michaelis Menton Plot Effect of Ki and I 200 180 Vmax No I Vo uM P m min 160 140 120 100 I 1 uM Ki uM Km app uM 10 11 1 20 0 1 110 80 60 40 Km 10 uM 20 0 0 50 100 150 200 Substrate uM Km app Km 1 I Ki Notice as Ki decreases the effect on Km app increases Why What are the values for Ki Km What does this tell you Similar plots could be made holding Ki constant and I Effect of Ki on LB Plot 0 07 Ki 1 uM Ki 10 uM 0 06 1 Vo 0 05 Ki 0 1 uM 0 04 No Inhibitor 0 03 0 02 slope Km Vmax 0 01 1 Vmax 0 0 1 1 Km 0 0 1 1 Km app s 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 S Notice as Km app increases 1 Km app decreases 1 Vmax is the same for all plots Enzymes Inhibition 6 BIOC 460 Summer 2010 Enzyme Inhibition A Painful Topic Since ancient times it has been known that chewing bark of pussy willow bushes would cure headaches and joint pain Late 1800 s 1800 s salicylic acid was isolated and identified as the active ingredient 1899 Bayer Chemical Company marketed a chemically modified form acetylsalicylate as Aspirin 1990 s super NSAIDS or COX 2 inhibitors marketed Celebrex Vioxx Bextra 2004 Vi 2004 Vioxx and d Bextra B t withdrawn ithd ffrom market k t 2009 Celebrex only is still on the market What are COX 1 and 2 What do they do COX cyclooxygenase aka prostaglandin H2 synthase External stimuli results in PLA2 activity free arachidonate COX is an ER enzyme that converts arachidonate to eicosanoids prostaglandins COX 1 produces PGH2 to regulate gastric mucin production constituitive COX 2 produces PGH2 in response to pain fever or inflammations inducible Thromboxanes produced in blood platelets initiate blood clotting Km for arachidonate is in low uM range Enzymes Inhibition 7 BIOC 460 Summer 2010 Aspirin acetylsalicylate the original non steroidal antiinflammatory drug NSAID See also Berg et al 6th ed Fig 12 25 p 339 Aspirin acetylates a …
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