Enzyme Mechanisms by HC12072707213

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									           Enzyme Mechanisms



                  Andy Howard
       Introductory Biochemistry, Fall 2008
            Thursday 23 October 2008

Biochemistry: Enzyme Mechanisms
                                  1       10/23/2008
 How do enzymes reduce
 activation energies?
  We want to understand what is
   really happening chemically when
   an enzyme does its job.
  We’d also like to know how
   biochemists probe these systems.


10/23/2008   Biochemistry: Enzyme Mechanisms   p. 2 of 63
    Mechanism Topics
   Inhibitors,                  Diffusion-controlled
    concluded:                    Reactions
    Pharmaceuticals              Binding Modes of
   Mechanisms:                   Catalysis
    Terminology                  Induced-fit
   Transition States            Tight Binding of
                                  Ionic Intermediates
   Enzyme chemistry
                                 Serine proteases



10/23/2008   Biochemistry: Enzyme Mechanisms    p. 3 of 63
   Most pharmaceuticals are
   enzyme inhibitors
    Some are inhibitors of enzymes that are
     necessary for functioning of pathogens
    Others are inhibitors of some protein
     whose inappropriate expression in a
     human causes a disease.
    Others are targeted at enzymes that are
     produced more energetically by tumors
     than they are by normal tissues.

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 4 of 63
   Characteristics of
   Pharmaceutical Inhibitors
     Usually competitive, i.e. they raise Km
      without affecting Vmax
     Some are mixed, i.e. Km up, Vmax down
     Iterative design work will decrease Ki
      from millimolar down to nanomolar
     Sometimes design work is purely blind
      HTS; other times, it’s structure-based

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 5 of 63
Amprenavir
   Competitive inhibitor of HIV
    protease,
    Ki = 0.6 nM for HIV-1
   No longer sold: mutual
    interference with rifabutin,
    which is an antibiotic used
    against a common HIV                            Quic kTim e™ and a
                                           TIFF (Uncompres sed) decompressor
                                              are needed to s ee this pic ture.
    secondary bacterial infection,
    Mycobacterium avium
10/23/2008   Biochemistry: Enzyme Mechanisms                p. 6 of 63
 When is a good inhibitor a
 good drug?
    It needs to be bioavailable and nontoxic
    Beautiful 20nM inhibitor is often neither
    Modest sacrifices of Ki in improving
     bioavailability and non-toxicity are okay if
     Ki is low enough when you start
     sacrificing


10/23/2008   Biochemistry: Enzyme Mechanisms   p. 7 of 63
     How do we lessen toxicity
     and improve bioavailability?
    Increase solubility…
     that often increases Ki because the van
     der Waals interactions diminish
    Solubility makes it easier to get the
     compound to travel through the
     bloodstream
    Toxicity is often associated with fat
     storage, which is more likely with
     insoluble compounds

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 8 of 63
     Drug-design timeline
         2 years of research, 8 years of trials


                    Preliminary toxicity testing
-3                                                                               100




                                                                                  Cost/yr, 106 $
-8                                                                                10
     Research                                         Clinical Trials
     0        2                                    Time, Yrs            10
 10/23/2008       Biochemistry: Enzyme Mechanisms                            p. 9 of 63
     Atomic-Level Mechanisms
    We want to understand atomic-level
     events during an enzymatically
     catalyzed reaction.
    Sometimes we want to find a way to
     inhibit an enzyme
    in other cases we're looking for more
     fundamental knowledge, viz. the ways
     that biological organisms employ
     chemistry and how enzymes make
     that chemistry possible.

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 10 of 63
 How we study mechanisms
     There are a variety of
      experimental tools available for
      understanding mechanisms,
      including isotopic labeling of
      substrates, structural methods,
      and spectroscopic kinetic
      techniques.


10/23/2008   Biochemistry: Enzyme Mechanisms   p. 11 of 63
    Ionic reactions
   Define them as reactions that involve
    charged, or at least polar, intermediates
   Typically 2 reactants
        Electron rich (nucleophilic) reactant
        Electron poor (electrophilic) reactant
   Conventional to describe reaction as
    attack of nucleophile on electrophile
   Drawn with nucleophile donating
    electron(s) to electrophile


10/23/2008     Biochemistry: Enzyme Mechanisms    p. 12 of 63
   Attack on Acyl Group
     Transfer of an acyl group: scheme 6.1
     Nucleophile Y attacks carbonyl carbon,
      forming tetrahedral intermediate
     X- is leaving group




10/23/2008   Biochemistry: Enzyme Mechanisms   p. 13 of 63
  Direct Displacement
     Attacking group adds to face of
      atom opposite to leaving group
      (scheme 6.2)
     Transition state has five ligands;
      inherently less stable than scheme
      6.1




10/23/2008   Biochemistry: Enzyme Mechanisms   p. 14 of 63
     Cleavage Reactions
    Both electrons stay with one atom
         Covalent bond produces carbanion:
          R3—C—H  R3—C:- + H+
         Covalent bond produces carbocation:
          R3—C—H  R3—C+ + :H-
    One electron stays with each product
         Both end up as radicals
         R1O—OR2  R1O• + •OR2
         Radicals are highly reactive—
          some more than others


10/23/2008     Biochemistry: Enzyme Mechanisms   p. 15 of 63
  Oxidation-Reduction
  Reactions
   Commonplace in biochemistry: EC 1
   Oxidation is a loss of electrons
   Reduction is the gain of electrons
   In practice, often:
        oxidation is decrease in # of C-H bonds;
        reduction is increase in # of C-H bonds
   Intermediate electron acceptors and donors are
    organic moieties or metals
   Ultimate electron acceptor in aerobic organisms
    is usually dioxygen (O2)

10/23/2008      Biochemistry: Enzyme Mechanisms     p. 16 of 63
     Biological redox reactions
   Generally 2-electron transformations
   Often involve alcohols, aldehydes, ketones,
    carboxylic acids, C=C bonds:
   R1R2CH-OH + X  R1R2C=O + XH2
   R1HC=O + X + OH- R1COO- + XH2
   X is usually NAD, NADP, FAD, FMN
   A few biological redox systems involve metal ions
    or Fe-S complexes
   Usually reduced compounds are higher-energy
    than the corresponding oxidized compounds


10/23/2008   Biochemistry: Enzyme Mechanisms   p. 17 of 63
               Overcoming the barrier
                   Simple system:
                    single high-energy transition state
                    intermediate between reactants,
                    products
Free Energy




                                  G‡
                           R
                                                 P

                            Reaction Coordinate
              10/23/2008   Biochemistry: Enzyme Mechanisms   p. 18 of 63
                       Intermediates
                  Often there is a quasi-stable intermediate
                   state midway between reactants &
                   products; transition states on either side
                                    T2
Free Energy




                           T1

                                I

                   R                            P

                       Reaction Coordinate
              10/23/2008    Biochemistry: Enzyme Mechanisms   p. 19 of 63
    Activation energy &
    temperature
   It’s intuitively sensible that higher
    temperatures would make it easier           Svante
    to overcome an activation barrier           Arrhenius

   Rate k(T) = Q0exp(-G‡/RT)
    G‡ = activation energy or
    Arrhenius energy
   This provides tool for measuring
    G‡

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 20 of 63
           Determining G‡
                                  Remember
                                   k(T) = Q0exp(-G‡/RT)
                                  ln k = lnQ0 - G‡/RT
                                  Measure reaction rate
                                   as function of
ln k                               temperature
                                  Plot ln k vs 1/T; slope
                                   will be -G‡/R


                            1/T, K-1
       10/23/2008   Biochemistry: Enzyme Mechanisms   p. 21 of 63
   How enzymes alter G‡
      Enzymes reduce G‡ by
       allowing the binding of the
       transition state into the active
       site
      Binding of the transition state
       needs to be tighter than the
       binding of either the reactants
       or the products.


10/23/2008    Biochemistry: Enzyme Mechanisms   p. 22 of 63
    G‡ and Entropy

   Effect is partly entropic:
   When a substrate binds,
    it loses a lot of entropy.
   Thus the entropic disadvantage of (say)
    a bimolecular reaction is soaked up in
    the process of binding the first of the
    two substrates into the enzyme's active
    site.

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 23 of 63
       Enthalpy and transition states
      Often an enthalpic component to
       the reduction in G‡ as well
      Ionic or hydrophobic interactions
       between the enzyme's active site
       residues and the components of
       the transition state make that
       transition state more stable.




10/23/2008   Biochemistry: Enzyme Mechanisms   p. 24 of 63
    Two ways to change G‡
   Reactants bound by           Transition state is
    enzyme are properly
                                  stabilized
    positioned
   Get into
    transition-state
    geometry more
    readily
    E A                           E A
       B                            B


    A+B                           A+B
                    A-B                              A-B

10/23/2008   Biochemistry: Enzyme Mechanisms     p. 25 of 63
   Reactive sidechains in a.a.’s
AA      Group         Charge Functions
                      @pH=7
Asp —COO-             -1     Cation binding, H+ transfer
Glu —COO-             -1     Same as above
His     Imidazole     ~0        Proton transfer
Cys —CH2SH            ~0        Covalent binding of acyl gps
Tyr Phenol            0         H-bonding to ligands
Lys NH3+              +1        Anion binding, H+ transfer
Arg     guanadinium
               +1               Anion binding
Ser     —CH2OH 0                See cys
10/23/2008    Biochemistry: Enzyme Mechanisms     p. 26 of 63
 Generalizations about active-
 site amino acids
    Typical enzyme has 2-6 key catalytic
     residues
    His, asp, arg, glu, lys account for 64%
    Remember:
         pKa values in proteins sometimes different
          from those of isolated aa’s
         Frequency overall  Frequency in catalysis


10/23/2008     Biochemistry: Enzyme Mechanisms   p. 27 of 63
    Cleavages by base
   Simple cleavage:

        —X—H + :B  —X:- + H—B+

     This works if X=N,O; sometimes C
     
 Removal of proton from H2O to cleave C-X:
   O                 O-          O

 —C—N        —C—N  —C—OH + HN

                       HO                           :B
    O                           H—B+
H        H :B

10/23/2008      Biochemistry: Enzyme Mechanisms   p. 28 of 63
      Cleavage by acid
     Covalent bond may break more easily if
      one of its atoms is protonated
     Formation of unstable intermediate,
      R-OH2+, accelerates the reaction
     Example:

             + OH-  R—OH  R—OH2+
                 (Slow)
      R+
      (Fast)
        R+ + H2O
10/23/2008     Biochemistry: Enzyme Mechanisms   p. 29 of 63
  Covalent catalysis
    Reactive side-chain can be a
     nucleophile or an electrophile, but
     nucleophile is more common
       A—X + E  X—E + A

       X—E + B  B—X + E

    Example: sucrose phosphorylase
         Net reaction:
          Sucrose + Pi  Glucose 1-P + fructose
         Fructose=A, Glucose=X, Phosphate=B


10/23/2008     Biochemistry: Enzyme Mechanisms   p. 30 of 63
    Rates often depend on pH
   If an amino acid that is necessary to
    the mechanism changes protonation
    state at a particular pH, then the
    reaction may be allowed or
    disallowed depending on pH
   Two ionizable residues means there
    may be a narrow pH optimum for
    catalysis



10/23/2008   Biochemistry: Enzyme Mechanisms   p. 31 of 63
                                 Papain as an example
                         1




                                           Papain pH-rate profile
relative reaction rate




                                       Cys-25                    His-159




                         0
                             2     3   4        5   6        7      8      9     10     11
                                                        pH




             10/23/2008                Biochemistry: Enzyme Mechanisms         p. 32 of 63
  Diffusion-controlled reactions
    Some enzymes are so efficient that the
     limiting factor in completion of the
     reaction is diffusion of the substrates
     into the active site:
    These are diffusion-controlled
     reactions.
    Ultra-high turnover rates: kcat ~ 109 s-1.
    We can describe kcat / Km as catalytic
     efficiency of an enzyme. A diffusion-
     controlled reaction will have a catalytic
     efficiency on the order of 108 M-1s-1.
10/23/2008   Biochemistry: Enzyme Mechanisms   p. 33 of 63
  Triosephosphate isomerase
  (TIM)
    dihydroxyacetone phosphate 
     glyceraldehyde-3-phosphate

                                     Glyc-3-P


             DHAP   
                                     Km=10µM
                                     kcat=4000s-1
                                     kcat/Km=4*108M-1s-1

10/23/2008     Biochemistry: Enzyme Mechanisms   p. 34 of 63
 TIM mechanism
     DHAP carbonyl H-bonds to neutral
      imidazole of his-95; proton moves from
      C1 to carboxylate of glu165
     Enediolate intermediate (C—O- on C2)
     Imidazolate (negative!) form of his95
      interacts with C1—O-H)
     glu165 donates proton back to C2
     See Fort’s treatment or fig. 6.7.

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 35 of 63
    Examining enzyme
    mechanisms will help us
    understand catalysis

   Examining general principles of catalytic
    activity and looking at specific cases will
    facilitate our appreciation of all enzymes



10/23/2008    Biochemistry: Enzyme Mechanisms   p. 36 of 63
       Binding modes:
       proximity
    We describe enzymatic mechanisms in terms            William
     of the binding modes of the substrates (or,          Jencks
     more properly, the transition-state species) to
     the enzyme.
    One of these involves the proximity effect,
     in which two (or more) substrates are
     directed down potential-energy gradients to
     positions where they are close to one
     another. Thus the enzyme is able to defeat
     the entropic difficulty of bringing substrates
     together.
    10/23/2008   Biochemistry: Enzyme Mechanisms   p. 37 of 63
   Binding modes: efficient
   transition-state binding
     Transition state fits even better
      (geometrically and electrostatically) in
      the active site than the substrate would.
      This improved fit lowers the energy of
      the transition-state system relative to
      the substrate.
     Best competitive inhibitors of an
      enzyme are those that resemble the
      transition state rather than the substrate
      or product.
10/23/2008   Biochemistry: Enzyme Mechanisms   p. 38 of 63
   Adenosine deaminase with
   transition-state analog
      Transition-state analog:
       Ki~10-8 * substrate Km
      Wilson et al (1991) Science 252: 1278



                    QuickTime™ and a
                TIFF (LZW) decompressor
             are neede d to see this picture.




10/23/2008   Biochemistry: Enzyme Mechanisms    p. 39 of 63
 Induced fit
     Refinement on original Emil
      Fischer lock-and-key notion:
     both the substrate (or transition-
      state) and the enzyme have
      flexibility
     Binding induces conformational
      changes



10/23/2008   Biochemistry: Enzyme Mechanisms   p. 40 of 63
     Example: hexokinase
    Glucose + ATP  Glucose-6-P + ADP
    Risk: unproductive reaction with water
    Enzyme exists in open & closed forms
    Glucose induces conversion to closed
     form; water can’t do that
    Energy expended moving to closed form




10/23/2008   Biochemistry: Enzyme Mechanisms   p. 41 of 63
    Hexokinase structure
   Diagram courtesy E. Marcotte, UT Austin




10/23/2008   Biochemistry: Enzyme Mechanisms   p. 42 of 63
 Tight binding of ionic
 intermediates
    Quasi-stable ionic species strongly bound
     by ion-pair and H-bond interactions
    Similar to notion that transition states are
     the most tightly bound species, but these
     are more stable



10/23/2008   Biochemistry: Enzyme Mechanisms   p. 43 of 63
   Serine protease mechanism

      Only detailed mechanism that we’ll ask
       you to memorize
      One of the first to be elucidated
      Well studied structurally
      Illustrates many other mechanisms
      Instance of convergent and divergent
       evolution

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 44 of 63
   The reaction
      Hydrolytic cleavage of peptide bond
      Enzyme usually works on esters too
      Found in eukaryotic digestive enzymes
       and in bacterial systems
      Widely-varying substrate specificities
            Some proteases are highly specific for
             particular aas at position 1, 2, -1, . . .
            Others are more promiscuous
         CH             NH
                                   C
  NH               C
             R1               CH       NH

10/23/2008         O                R-1
                  Biochemistry: Enzyme Mechanisms   p. 45 of 63
 Mechanism
    Active-site serine —OH …
     Without neighboring amino acids, it’s fairly
     non-reactive
    becomes powerful nucleophile because OH
     proton lies near unprotonated N of His
    This N can abstract the hydrogen at near-
     neutral pH
    Resulting + charge on His is stabilized by its
     proximity to a nearby carboxylate group on
     an aspartate side-chain.


10/23/2008    Biochemistry: Enzyme Mechanisms   p. 46 of 63
 Catalytic triad
   The catalytic triad of asp, his, and ser is
    found in an approximately linear
    arrangement in all the serine proteases,
    all the way from non-specific, secreted
    bacterial proteases to highly regulated
    and highly specific mammalian
    proteases.



10/23/2008   Biochemistry: Enzyme Mechanisms   p. 47 of 63
 Diagram of first three steps




10/23/2008   Biochemistry: Enzyme Mechanisms   p. 48 of 63
    Diagram of last four steps




                            Diagrams courtesy
                            University of Virginia
10/23/2008   Biochemistry: Enzyme Mechanisms   p. 49 of 63
    Chymotrypsin as example
   Catalytic Ser is Ser195
   Asp is 102, His is 57
   Note symmetry of mechanism:
    steps read similarly L R and R  L




              Diagram courtesy of
              Anthony Serianni,
              University of Notre Dame
10/23/2008   Biochemistry: Enzyme Mechanisms   p. 50 of 63
      Oxyanion hole
   When his-57 accepts proton from Ser-195:
    it creates an R—O- ion on Ser sidechain
   In reality the Ser O immediately becomes
    covalently bonded to substrate carbonyl carbon,
    moving - charge to the carbonyl O.
   Oxyanion is on the substrate's oxygen
   Oxyanion stabilized by additional interaction in
    addition to the protonated his 57:
    main-chain NH group from gly 193 H-bonds to
    oxygen atom (or ion) from the substrate,
    further stabilizing the ion.

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 51 of 63
      Oxyanion
      hole cartoon


   Cartoon courtesy
    Henry Jakubowski,
    College of
    St.Benedict /
    St.John’s University

      10/23/2008   Biochemistry: Enzyme Mechanisms   p. 52 of 63
 Modes of catalysis in serine
 proteases
    Proximity effect: gathering of reactants in steps
     1 and 4
    Acid-base catalysis at histidine in steps 2 and 4
    Covalent catalysis on serine hydroxymethyl
     group in steps 2-5
    So both chemical (acid-base & covalent) and
     binding modes (proximity & transition-state) are
     used in this mechanism

10/23/2008    Biochemistry: Enzyme Mechanisms   p. 53 of 63
    Specificity
   Active site catalytic triad is nearly invariant for
    eukaryotic serine proteases
   Remainder of cavity where reaction occurs
    varies significantly from protease to protease.
   In chymotrypsin  hydrophobic pocket just
    upstream of the position where scissile bond sits
   This accommodates large hydrophobic side
    chain like that of phe, and doesn’t comfortably
    accommodate hydrophilic or small side chain.
   Thus specificity is conferred by the shape and
    electrostatic character of the site.

10/23/2008    Biochemistry: Enzyme Mechanisms   p. 54 of 63
 Chymotrypsin active site
    Comfortably
     accommodates
     aromatics at S1 site
    Differs from other
     mammalian serine
     proteases in specificity
Diagram courtesy School
of Crystallography,
Birkbeck College

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 55 of 63
 Divergent evolution
    Ancestral eukaryotic serine proteases
     presumably have differentiated into forms
     with different side-chain specificities
    Chymotrypsin is substantially conserved
     within eukaryotes, but is distinctly
     different from elastase




10/23/2008   Biochemistry: Enzyme Mechanisms   p. 56 of 63
     iClicker quiz!
    Why would the nonproductive hexokinase
     reaction H2O + ATP -> ADP + Pi
     be considered nonproductive?
    (a) Because it needlessly soaks up water
    (b) Because the enzyme undergoes a wasteful
     conformational change
    (c) Because the energy in the high-energy
     phosphate bond is unavailable for other
     purposes
    (d) Because ADP is poisonous
    (e) None of the above

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 57 of 63
 iClicker quiz, question 2:
 Why are proteases often
 synthesized as zymogens?
    (a) Because the transcriptional machinery
     cannot function otherwise
    (b) To prevent the enzyme from cleaving
     peptide bonds outside of its intended realm
    (c) To exert control over the proteolytic reaction
    (d) None of the above


10/23/2008    Biochemistry: Enzyme Mechanisms   p. 58 of 63
 Question 3: what would bind
 tightest in the TIM active site?
    (a) DHAP (substrate)
    (b) D-glyceraldehyde (product)
    (c) 2-phosphoglycolate
     (Transition-state analog)
    (d) They would all bind equally well



10/23/2008   Biochemistry: Enzyme Mechanisms   p. 59 of 63
    Convergent evolution
   Reappearance of ser-his-asp triad in
    unrelated settings
   Subtilisin: externals very different from
    mammalian serine proteases; triad same




10/23/2008   Biochemistry: Enzyme Mechanisms   p. 60 of 63
    Subtilisin mutagenesis
   Substitutions for any of the amino acids in the
    catalytic triad has disastrous effects on the
    catalytic activity, as measured by kcat.
   Km affected only slightly, since the structure of
    the binding pocket is not altered very much by
    conservative mutations.
   An interesting (and somewhat non-intuitive)
    result is that even these "broken" enzymes
    still catalyze the hydrolysis of some test
    substrates at much higher rates than buffer
    alone would provide. I would encourage you
    to think about why that might be true.

10/23/2008   Biochemistry: Enzyme Mechanisms   p. 61 of 63
          Cysteinyl proteases
    Ancestrally related to ser
     proteases?
    Cathepsins, caspases,
     papain
    Contrasts:
         Cys —SH is more basic
          than ser —OH
         Residue is less hydrophilic
         S- is a weaker nucleophile      Diagram courtesy of
          than O-                         Mariusz Jaskolski,
                                          U. Poznan

    10/23/2008      Biochemistry: Enzyme Mechanisms   p. 62 of 63
       Papain active site




Diagram courtesy
Martin Harrison,
Manchester University

     10/23/2008   Biochemistry: Enzyme Mechanisms   p. 63 of 63

								
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