Experimental Design. Margaret A. Daugherty. Fall Michaelis Menton Kinetics and Inhibition. Lecture 14: Enzymes & Kinetics III E + S ES E + P

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1 Lecture 14: Enzymes & Kinetics III Michaelis Menton Kinetics and Inhibition Margaret A. Daugherty Fall 2003 Experimental Design k 1 k cat E + S ES E + P k -1 I want to measure the reactivity of my enzyme how? Measure production of [P] Measure the decrease of [S] BIC 205

2 Experimental Design Measure production of [P] otes: (1). Must be done at constant [E] because V max = k cat [E] t (2). The velocity of an enzyme-catalyzed reaction is dependent upon the substrate concentration [S] (3). Substrate saturation occurs. (4). ther variables to hold constant: T, p and ionic strength. Expt must have... BIC 205 The problem: we can t use substrate saturation curves directly to obtain Vmax and Km V max To reach a v o =.99Vmax; we need [S] = 99 x [Km]

3 V = V max[s] K M + [S] Lineweaver-Burke Plot v vs. [S] 1/v vs 1/[S] 1 V K M = V max [S] y = mx + b V max Extrapolate BIC 205 utline Inihibition Kinetics Reversible competitive non-competitive Irreversible Distinguishable by kinetic inhibition patterns

4 Enzyme Inhibition Inhibitor (I): compound that decreases the rate of a catalyzed reaction Two modes of action: Reversible: I interacts via non-covalent interactions with E Irreversible: I interacts via covalent interactions with E (termed suicide inhibitors) Reversible Inhibition Inhibitors interact with the enzymes through noncovalent interactions. They can easily associate and dissociate from the enzyme. Two general classes (although more exist!): Competitive Inhibition: I binds to same site as S oncompetitive Inhibition: I binds to different site than S

5 Competitive Inhibition I mimics S for binding; I reversibly binds into active site; Binding of I prevents S from binding owever, increasing S can compete off I Results in an apparent increase in K M for S substrate inhibitor Competitive Inhibition Michaelis-Menton Equation [E] t = [E] + [ES] + [EI] V = Vmax [S] K M (1+[I]/K I ) + [S] V = Vmax [S] K M app + [S]

6 V vs. [ S ] Graph for Competitive Inhibition Competitive inhibitors alter K m but do not change V max [ I ] = [ I ] = 1 velocity [ I ] = [ S ] Identification of Competitive Inhibition In the presence of a competitive inhibitor K M increase; V max remains the same; Slope = K M /V max ; Slope increases with [I] Inhibition can be overcome by increasing [S] - hence competitive!

7 Competitive inhibitors are not cures: IV/AIDS TERAPY 2002 Cumulative umber of AIDS deaths orth America 15, ,000 World 3,100,000 24,900,000 Total # of infected people orth America 980,000 1,400,000 World 42,000,000 61,800,000 # of new infections orth America 45,000 World 5,000,000 umber of people cured of IV <1 umber of infections prevented by vaccination <1 * UAIDS, December 2002 IV Infection Kills helper T cells; B cells can t efficiently proliferate in response to antigen stimulation.

8 Viral efficiency: viral proteins synthesized as large poly-protein Role of IV protease Excise itself Mature stuctural proteins IV Protease Characteristics Aspartyl protease (pepsin family); omodimer, 99AAs/monomer; All β structure; Active site Asp from each monomer Kd = 440 nm; kcat = 6.8 / sec (pepsin 400/sec) Substrates ydrophobic, unbranched AAs at the terminal side of the bond to be cleaved; Small peptides (7 AAs). Wlodawer 2002

9 Competitive Inhibitors - IV protease inhibitors Ritonavir and saquinavir are used to treat IV / AIDS. Inhibits IV protease (a aspartic protease) which cleaves larger viral protein precursors into IV proteins needed for the virus particle. R Phe Pro R' S Ritonavir S 2 Saquinavir V vs. [ S ] Graph for Competitive Inhibition Problem 1: inhibitors bind reversibly! Competitive inhibitors alter K m but do not change V max [ I ] = 0 velocity [ I ] = 1 [ I ] = [ S ]

10 Ritonavir S S K i ~ 15 pm; igh (> 10 5 ) specificity for IV protease over pepsin; 70% bio-availability Good absorption t 1/2 ~ 1.5 hr Problem 2: Inhibitor Clearance The aspect of clearance proved to be the biggest challenge: drug metabolism is carried out by a number of enzymes including P450 (cyctochrome based oxidising enzymes) Structure modifying enzymes (e.g glutathione S-transferases and various acetyl transferases) Ritonavir was found to remain available as a result of being a specific and potent (not to mention fortuitous) inhibitor of cytochrome p450 Martin 2000

11 Problem 3: IV Mutation Rate Gives rise to Drug Resistance 3.4 x 10-5 IV-1 mutations/base/replication event; IV-1 replications/day; Thus, 340,000 mutations/base/day ow do we control something that is changing so rapidly? Triple Cocktails 2002 IV Protease inhibitor Amprenavir Indinavir Lopinavir efinavir Ritonavir Saquinavir IV RT inhibitors (non-nucleoside analogues, RTI) Delavirdine Efavirenz evirapine IV RT inhibitors (nucleoside analogues, RTI) Abacavir Didanosine (ddi) Lamivudine (3TC) Stavudine (d4t) Tenofovir Zalcitabine (ddc) Zidovudine (AZT)

12 Anti-IV Therapies Successful: ucleoside (nucleotide) RT inhibitors onnucleoside RT inhibitors Protease inhibitors Promising: Fusion inhibitors Integrase inhibitors Unsuccessful (so far): Immunotherapy Gene therapy Recombinant antiviral proteins erbal extracts Faith healing Wlodawer 2002 on-competitive Inhibition I binds to a site other than the active site Does not compete with S for binding to its site. K M remains unchanged. I can bind to both E and ES I prevents catalysis from occurring. V max is altered I can not be competed off by increasing [S] Chem 204

13 on-competitive Inhibition Michaelis-Menton Equation k cat app = k cat /(1+[I]/K I ) et result: enzyme becomes less effective in catalyzing chemical reactions V app max = k app cat [E] t V app V = max [S] K M + [S] V vs. [ S ] Graph for on-competitive Inhibition Binding of S to E is unchanged ---> K M unchanged Presence of I affects V max ---> Vmax decreases

14 Identification of oncompetitive Inhibition In the presence of a noncompetitive inhibitor K M is unchanged; V max decreases; The effects of noncompetitive inhibition cannot be overcome by increasing [S]. ne step further - mixed non-competitive inhibition I can bind to E or ES; Affinity differs for E and ES

15 Irreversible Inhibition Inhibitors interact with the enzymes through covalent attachment of the inhibitor to the enzyme; The formation of the covalent bond is a slow process. A time-dependent decrease in enzymatic activity is observed; Derivatized enzyme is no longer functional. Example of Irreversible Inhibition rganophosphates are used in insecticides and nerve gases Irreversible inhibitors of acetylcholinesterase React with the active site serine LD 50 of sarin in humans is ~ 0.01mg/kg!

16 Affinity Labels Inhibitor contains a chemically reactive functional group which forms a covalent bond between the enzyme and inhibitor. k 1 E + I E I E I k 2 k -1 where k 2 = k inact or k app Ly s (C 2)4 Ly s (C 2)4 2 R R C C2 B r C C2 Time-dependent Decrease in Enzyme Activity v i t c a g n i n i a m e r % Decrease in enzyme activity over time [ I ] v i t c a g n i n i a m e r % Protection by substrate Constant [ I ] [ S ] time time

17 Penicillin - an affinity inhibitor R penicillin S C R S C C 2 Ser glycopeptide transpeptidase C 2 Ser penicillin-enzyme complex Suicide Inhibitors as Drugs Used to inactivate the enzyme; Relatively unreactive until bind to active site; Very reactive when combine irreversibly with the enzyme; Rational drug design; Very effective and few side effects.

18 Review 1). Enzymes can be either reversibly or irreversibly inhibited. 2). An inhibitor is a compound that decreases that rate of a catalyzed reaction. 3). Reversible inhibition: (know the effects on the substrate saturation curves Lineweaver-Burke plot) Competitive: V max unaltered; K m (K m app ) increased; Substrate competes for or blocks substrate binding site. on-competitive inhibition: V max (V max app decreased (hence ability to catalyze reaction affected); K m unaltered. 4). Irreversible inhibition occurs when the inhibitor binds irreversibly (covalently) to the active site, thus completely preventing the substrate from having access to it.