Class Note for BIOC 460 at UA 2
Class Note for BIOC 460 at UA 2
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Date Created: 02/06/15
BIOC 460 Spring 2008 Lecture 12 Enzymes Inhibition Reading Berg Tymoczko amp Stryer 6th ed Chapter 8 pp 225236 Problems pp 238239 chapter 8 1 2 4ab 5ab 7 10 Jmol structure cyclooxygenaselnonsteroidal antiin ammatory drugs into HWva biocnem arizona educiasseshioc462462airnoicox iZcoxi i iitm Key Concepts Km and me can be determined from double reciprocalplots 1Nn vs 1lS Enzyme inhibitors compounds that reduce velocity of enzymecatalyzed reactions Ztypes revers reversible competIt Inhibitor I increases Km but has no effect on me uncompetitive I decreases both Km and me by same factor pure noncompetitive I decreases mm but has no effect on Km can distinguish different types of reversible inhibitors using double reciprocal plots 1ND vs 1S in absence ofl and in presence of different concentrations of Irreversible in bitors cause irreversible generally covalent modi cation ofthe enzyme inactivating it several types groupspeci c chemical modifying reagents that would react with certain types of functional groups on many different enzymes substrate analogs with a reactive group on them so more speci c for one enzyme quotsuicidequot substrates mechanismbased inhibitors not reactive until the speci c chemical mechanism oftheir target enzyme makes them quotkillquot covalently modify the active site they39re in le and irreversible 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 info about control ofmetabolic pathways design ofdrugs Learning Objectives Terminology double reciprocal plot reversible inhibition competitive pure noncompetitive uncompetitive irreversible inhibition affinity label transition state analog suicide inhibition mechanismbased inhibitor Given a hyperbolic VNalt vs S plot explain the meaning ofthe ratio anszX in terms of occupied active site concentration and total active site concentration and nd Km directly from the graph including its units Given a LineweaverBurk plot 1ND vs 1lS ndcalculate Vmax and Km from the graph Learning Objectives continued Explain competitive uncompetitive and pure noncompetitive inhibition in terms ofa diagram of linked reaction equilibria forformation of E8 El and if it can form EIS See Berg et al Figs 817 818 819 Explain how these 3 types of inhibition can be distinguished from each other graphically a on a VB vs S plot and b on a double reciprocal LineweaverBurk plot What is the effect of a competitive inhibitor on Km and on Vmax compared to the values in absence of inhibitor What is the effect of an uncompetitive inhibitor on Km and on Vma compared to the values in absence of inhibitor What is the effect of a pure noncompetitive inhibitor on Km and on Vmax compared to the values in absence of inhibitor Why is ethanol used as an antidote for ethylene glycol antifreeze poisoning What type of inhibitor is pencillin Give some other speci c examples of reversible and irreversible enzyme inhibition Graphical Determination of Kquot and me Enzyme kinetics experiments measuring VD as a function of S to determine Vma and Km can use computer with programs to t data to MichaelisMenten equation extracting Km and Vma from the data can39t extrapolate quotby handquot on MichaelisMenten VB vs S hyperbolic plot to get accurate Vmax and Km values VV i mameISl simple solution without a computer use linear version double reciprocal plot LineweaverBurk Plot Take reciprocal of both sides of MM Equation and rearrange to get LineweaverBurk equation 1Km1 1 Va Vmax S Vmax Lineweaver Burk Plot double reciprocal plot LineweaverBurk Plot double reciprocal plot 1Nn vs 1s 1 V Ki 1 V 17 Va Vmax S Vmax Equation ofa straight line y mx b x intercept 1le 1lV0 Slope KMlea NOTE xintercept is a negative number which itself is the negative of the reciprocal of Km Km is always a posi ve number the substrate concentration at which v 12 vm yintercept 1me a slope Km lex Intercept I l KM Intercept 1me 15 Berg eiai Fig 8773 LEC 12 Enzymes Inhibition BIOC 460 Spring 2008 Lineweaver Burk Plot double reciprocal plot Enzyme Inhibitors L Km 1 7 Lineweaver3urk p t reversible rapid bindingrelease from enzyme in an equilibrium or Vo Vmax S Vmax double reciprocal plot irreversible very tightly bound to enzyme either covalently or 1No vs 1IS noncovalently but effectively don39t come off Equation ofa straight line y mx b Reversible inhibitors type of inhibition diagnosed by effect of inhibitor on Km and Vmax effectsquotdiagnosisquot obvious on double reciprocal plot basis for drugs39 actions research tools in figuring out enzyme chemical catalytic mechanisms x intercept 1IKm yintercept 1Nmax slope Km IVmax Slope KMIVmax 1vo What if xintercept 2 x 104 M 1 a reciprocal concentration extrapolated back Intercept 1KM to negative values to get intercept 1IKm 2x104M1 Reversible Inhibition competitive uncompetitive or noncompetitive defined operationally by their effects on enzyme kinetic parameters Km andoeraX Intercept 1Vm WhatisKm Km 1l 2x104M1 05x104Mor5x105M 2 1 0 1 1S Note POSITIVE value Can Kquot ever have a negative value ES compleEeversible Enzyme continued Competitive Inhibition Substrate inhibitor Berg er al Fig 815 Enzyme can bind either substrate or inhibitor but not both Either Inhibitor binds in same site as S or more rarely inhibitor binds to different site causing conformational change in active site so substrate can39t bind Enzyme active site can be free neither ligand bound E or have 5 Prevents S from binding so increases Km Egggdefs complex or have I bound El complex butthere s no ESI H no effecton Vmax Substrate Uncompetitive S inhibitor quotPurequot SUbS I ate Competitive inhibitor Noncompetitive increases apparent Km E gt E P Inhlb of but doesn39t affect Vmax a High 5 overcomes I u effect of I Decreases Km and reduces Vmax Ki by same factor so slope Has no effect on Km 5 of 1NO vs 1S doesn39t change no effect on 8 binding El 39 blnds only to ES complex reduces Vmax reduces km Berg et al Fig 817 Competitive Inhibition continued Uncompetitive Inhibition Competitive inhibitor increases apparent Km but doesn39t affect Vmax High 5 overcomes effect of I Inhibitor binds only to ES complex Binding site for l is created only upon 8 binding 100 N 39quoth39bquot 39 Km higherwith inhibitorthan There39s no detectible El complex only E E8 and ESI but ESI can39t 30 without inhibitor make product 39 Vmax quot01 Changed High 5 does NOT overcome effect of I 60 I 10Ki Competitive s Inhibitor EI gtESI gtEP No inhibitor Ki present ESI gt 40 Relative rate l5Ki 20 UV Substrate gt Berg et al Fig 817 Berg et al Fig 820 Berg et al Fig 818 0 15 LEC 12 Enzymes Inhibition BIOC 460 Spring 2008 Uncompetitive Inhibition continued pure Noncompetitive Inhibition Inhibitor binds only to ES complex 39 Binding Site f0 is created oniy upon 5 binding Enzyme can bind both substrate and inhibitor simultaneously but ESI There s no El complex only E E8 and ESI but ESI can t make product compex can39t make product 10 Noinhibimr Km Iowerthan without inhibitor Inhibitor must dissociate in order for catalysis to occur so 39Vmax i Wer by email same faCtOI Inhibitor binding decreases Vmax decreases kw but has no effect on Km w Slope of 1No vs 1IS unchanged 5 50 Un competitive 3 inhibitor 2 40 S R 2 Noinhibitor E I 4 ES gt E P KM for uninhibited enzyme S KsrpforillKl El 4 ESI gt Berg et al Fig 818 present Substrate V Berg et al Fig 819 Berg et al Fig 821 0 1IS Pure NoncompetitiVe iNhibitioni CONtinued Examples of reversible inhibitors competitive Enzyme can bind both substrate and inhibitor simultaneously ESI complex can39t make product so I must dissociate in orderfor 39 inh39b39mrs f Noinhibitor bios nthesis new 39 n effeCt n Km nucleic acids 80 High 8 does NOT overcome required for cell H H N N N catalysis to occur g gylggglate 1 Y I Inhibitor bindin decreases V decreases k 9 ma cat enzyme in HM 100 Vmax decreased nucleotide N 0 3 effect of I division 2 so N nmm emive vertebrate DHFR A quot inhibited by Diliydroinlnle Inhibitor 1 4 methotrexate H N N N 1 g selectively kills Y 1IV rapidly dividing cells 20 K cancer chemo N N No 39nh39b39mr prokaryotic DHFR 0 Present inhibited by quot1 sub tratei trimethoprimhas quot3C Berg etal Fig 819 me effecquot 0 human DHFR so a Berg etal Fig 822 0 ms 900d antibiotic B t I F 8 16 Meihofrexuie erg e a ig Another example of a reversible inhibitor competitive quotrevers39bie inhibition 1 groupspecific covalent modifying agents 2 affinity labels SUCCinat dehydr genase 3 transition state analogs enzyme In Cltrate cycle 4 suicide inhibitors mechanismbased inhibitors 1 group speci c covalent modifying agents react with specific type of 3ucb si39r ee compg lfvgaf hlbnon enzyme functional group eg SerOH or CysSH or His imidazole on w enzymeprotein Diisopropylphosphofluoridate DIPF potent nerve gas poison 0 0 0 reacts with specific CH 0 reactive Ser OH on 6 quot 2quot 1 0 0 0 many enzymes example reaction 0 0 With reactive 0H o P catalytic OH Ser o P p w group of acetyl 0 39 cholinesterase at synaptic H CH junctions CH 3 H a 3 modified enzyme more examples at the end of notes discussion of some examples of inactive pharmaceutically important enzyme inhibitors muykhulim mquot Inm39iw39ed Berg et al Fig 823 esteruse enzyme LEC 12 Enzymes Inhibition BIOC 460 Spring 2008 2 affinity labels structural similarity to substrate quotguidesquot reagent to active site reaction at active site covalently inactivates enzyme Example Tosyl phenylalanyl chloromethylketone TPCK hymntrypsin phenyl group binds in substrate specificity site of chymotrypsin H H Hi557 RyNCNRquot N H II 0 Natural svbsIrnh lor hymnllypsin TPCK Spetiiitity group 1 Berg et al Fig 825 Hc c Tosyllpllenylulunine thlnrnmelhyl kelone llPCKl L 3 transition state analogs structurally similar to transition state which binds even more tightly to enzyme than substrate binds so very high affinity for active site See Berg et al Fig 8 28 for an example Transition state analogs useful for understanding catalytic mechanisms clues about structure of transition state very specific inhibitors of enzymes pharmaceutical applications antigens for immunizing lab animals to generate antibodies with binding sites complementary to the transition state such that the antibodies themselves have catalytic activity quotabzymesquot 4 suicide substrates mechanismbased inhibitors Structural similarity to substrate quotguidesquot reagent to active site Enzyme treats it as asubstrate startin chemical catal ic rocess with inhibitor Chemical mechanism itself leads enzyme to react covalently with inhibitor thus quotcommitting suicide Mechanismbased inhibition depends on chemical mechanism of catalysis Example penicillin inhibits an enzyme a transpeptidase required for bacterial cell wall synthesis see below and text pp 232234 9 Just a few of thousands of pharmaceutically important enzyme inhibitors 1 Penicillin an antibiotic both a transitionstate analog and a suicide substrate covalently inhibits a transpeptidase enzyme involved in bacterial cell wall synthesis eukaryotic cells don39t have this enzyme Normal transpeptidase catalytic mechanism nucleophilic attack of enzyme Ser OH on substrate making a covalent acyIenzyme intermediate Covalent intermediate continues in enzymecatalyzed reaction to form peptide crosslink in peptidoglycan structure of cell wall regenerating free enzyme for another round of catalysis Penicillin resembles transition state in structure so penicillin a binds very tightly and b is very reactive Normal catalytic mechanism makes covalent intermediate with penicillin but enzymepenicillin derivative can39t continue Inhibitor is quotstuckquot on enzyme covalently attached and modified enzyme is now inactive because of its own catalytic activity it committed suicide See Berg et al 6th ed pp 232234 if you want a more thorough discussion of penicillin and its action more pharmaceutically important enzyme inhibitors 2 Aspirin acetylsalicylate a nonsteroidal antiinflammatory drug NSAID See also Berg et al 6th ed Fig 12 25 p 339 covalently irreversibly inactivates enzyme PGH2 synthase cyclooxygenase activity also known in its two forms as COX 1 and COX 2 involved in prostaglandin biosynthesis antiinflammatory action clue to blocking of prostaglandin synthesis covalently modifies acetylates specific SerOH group in channel through which substrate arachidonic acid a 20 C fatty acid must pass to reach active site NSA Ds block active site access inhibiting enzyme preventing prostaglandin synthesis reducing inflammation Jmol structure of COX 2 with various inhibitorsdrugs WW 0 H Acetylated 5e residue Aspirin also reduces blood clotting because same 0 cu 0 CH3 enzyme is also needed T 597530 T for synthesis of o thromboxane A2 TXAZ 0 involved in blood platelet ASEIIIIII aggregation in clotting Atelylsulltytllt and Berg et al Fig 1225 more pharmaceutically important enzyme inhibitors 3 other NSAIDs nonsteroidal antiinflammatory agents besides aspirin eg ibuprofen Motrin Advil acetaminophen Tylenol indomethacin naproxen Aleve competitive reversible inhibitors of cyclooxygenase activity of PGH2 synthase block prostaglandin synthesis and thus act as antiinflammatory agents reversibly bind in channel through which substrate must access enzyme active site so act as competitive inhibitors by preventing substrate binding even though they don t bind IN the active site Aspirin inhibits same enzyme irreversibly by acetylating Ser OH group in quotentrancequot channel to active site but not actually in active site Jmol structure of COX 2 with various inhibitorsdrugs 4 statins lnhibitors of HMGCoA reductase the ratelimiting control enzyme in cholesterol biosynthesis Competitive inhibitors of HMGCoA reductase are cholesterollowering drugs decrease rate of cellular cholesterol biosynthesis structures similar to substrate for HMGCoA reductase mevalonate eg Mevacor Iovastatin Pravachol pravastatin and Zocor simvastatin more pharmaceutically important enzyme inhibitors 5 Ethanol antidote for ethylene glycol antifreeze or methanol wood alcohol poisoning Toxic effects of ethylene glycol and of methanol depend on their OH groups being oxidized to aldehyde by alcohol dehydrogenase in body and then to carboxylic acids Ethanol another substrate with less toxic oxidation products competes for binding to alcohol dehydrogenase If alcohol dehydrogenase molecules are all occupied with ethanol as a substrate ethylene glycol or methanol passes through body without being oxidized and is excreted kidneys ethanol ethylene glycol substrate Inhibitor competing substrate H Ho H 6 antiHIV drugs antiAIDS AZT metabolized to AZTtriphosphate which terminates growing DNA chains in reaction catalyzed by HIV viral reverse transcriptase much higher affinity for HIV reverse transcriptase than for cellular DNA polymerases Saquinavir and Ritonavir VERY tightbinding inhibitors transition state analogs of HIV protease enzyme needed to process large HIV polyprotein precursors to release viral proteins LEC 12 Enzymes Inhibition
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