Enzyme Mechanisms

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Reading - Chapter 8
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Key Concepts

  • Mechanisms used by enzymes to enhance reaction rates include:
    • (1st 4 mechanisms based on BINDING of substrate and/or transition state)
      • Proximity & orientation
      • Electrostatic catalysis
      • Preferential binding of the transition state
      • Induced fit
    • General acid/base catalysis
    • Covalent (nucleophilic) catalysis
    • Metal ion catalysis

  • Under physiological conditions, the maximum rate per active site is achieved by maximizing kcat/Km AND having Km greater than the physiological [S]. 
    • This means that most of the enzyme will be free to interact with a substrate molecule and that every encounter will lead to a reaction.
    • However, some regulated enzymes haven't evolved for maximum rate per active site or for maximum efficiency -- they're better regulated with a lower Km value.

  • The chemical mechanism of serine proteases like chymotrypsin illustrates
    • not only proximity and orientation, but also:
      • Transition state stabilization.
      • Covalent catalysis, involving a catalytic triad of Asp, His and Ser in the active site.
      • general acid-base catalysis
      • electrostatic catalysis

  • The chemical mechanism of triose phosphate isomerase illustrates
    • Transition state stabilization
    • Acid-base catalysis
    • Electrostatic catalysis

  • Kinetic mechanism: order in which substrates (and products) bind and release from enzyme, kinetically identifiable intermediates formed (e.g., ES), including whatever kinetic steps can be identified, any known rate constants, etc.
  • Chemical mechanism: chemical pathway of conversion of S --> P, including structures of intermediates, what enzyme catalytic groups participate and chemically how they participate, etc.

Enzyme Catalytic Mechanisms

  • Enzymes use a variety of mechanisms to enhance reactions rates (lower free energies of activation).
  • There are 7 listed here, but some of the concepts overlap -- these "pigeonholes" may not be the only way to make a list.
  • Any one particular enzyme may use only a few of these mechanisms; some enzymes use many of them.

  • The first 4 are based on the BINDING of substrate and/or transition state, so reaction takes place in the active site, not in bulk solution. Enzyme is using binding energy to increase rate of reaction.

    1.  Proximity and orientation effects (sometimes referred to as "entropy reduction"):

    • Proximity: Reaction between bound molecules doesn't require an improbable collision of 2 molecules -- they're already in "contact" (increases the local concentration of reactants).
    • Orientation: Reactants are not only near each other on enzyme, they're oriented in optimal position to react, so the improbability of colliding in correct orientation is taken care of.
      Fig. 8-7 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): Rate enhancement by entropy reduction.
      a) bimolecular reaction (high activation energy, low rate)
      b) unimolecular reaction, rate enhanced by factor of 105 due to increased probability of collision/reaction of the 2 groups
      c) constraint of structure to orient groups better (elimination of freedom of rotation around bonds between reactive groups), rate enhanced by another factor of 103, for 108 total rate enhancement over bimolecular reaction.

    2. Electrostatic catalysis:

    • When substrate binds to enzyme, water is usually excluded from active site (desolvation).
      • causes local dielectric constant to be lower, which enhances electrostatic interactions in the active site, and also
      • results in protection of reactive groups from water, so water doesn't react to form unwanted biproducts.
        • Of course, if water is a substrate, it has to be "allowed in", but maybe only in a certain sub-part of active site.
    • Involvement of charged enzyme functional groups in stabilizing otherwise unstable intermediates in the chemical mechanism can also correctly be called "electrostatic catalysis".

    3.   Preferential transition state binding/transition state stabilization (formerly sometimes called "strain and distortion"):
    • probably the most important rate enhancing mechanism available to enzymes
    • Enzyme binds transition state of the reaction more tightly than either the substrate or product --
      therefore DG is reduced, and rate is enhanced
      .
    • Strain or stress?
      • "Strain" is a classic concept in which it was supposed that binding of the substrate to the enzyme somehow caused the substrate to become distorted toward the transition state.
        • It's unlikely that there is enough energy available in substrate binding to actually distort the substrate toward the transition state.
        • It's possible that the substrate and enzyme interact unfavorably and this unfavorable interaction is relieved in the transition state.
        • It's more likely that the enzyme is strained, as for example in induced fit (below). 
      • Transition state stabilization is a more modern concept: it is not the substrate that is distorted but rather that the transition state makes better contacts with the enzyme than the substrate does, so the full binding energy is not achieved until the transition state is reached.
      • Induced fit assumes that the active site of an enzyme is not complementary to that of the transition state in the absence of the substrate. Such enzymes will have a lower value of kcat/Km, because some of the binding energy must be used to support the conformational change in the enzyme. Induced fit increases Km without increasing kcat

    • Weak interactions between the enzyme and substrate are optimized in the transition state
      • See Fig. 8-5 below ("stickase" that catalyzes breakage of metal bar)
    • DG for formation of transition state is partially "paid for" by the energetics of weak interactions between enzyme and transition state.
    • BINDING of substrates and intermediates in the enzyme active site:
      • complementarity (chemical and shape complementarity) between enzyme and substrate/intermediates/transition state(s) has to be "fine-tuned" -- simply binding substrate tightly doesn't translate into better catalysis.

      • Fig. 8-5 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): Imaginary enzyme ("stickase") designed to catalyze "cleavage" (breaking) of a metal stick ("magnetic" interactions, red dashed lines, represent noncovalent interactions between enzyme and substrate and between enzyme and transition state ). Metal stick must be bent, a "high energy state", before it can be broken, so "transition state" is bent stick.

        a) no enzyme

        b) Enzyme-"catalyzed" reaction in which enzyme is highly complementary to substrate and NOT complementary to transition state, so free energy of ES complex is much lower and free energy of TS (‡) doesn't change
        Result: activation energy (DG) is much higher than in absence of "catalyst", so rate constant would be dramatically decreased

        c) Active site is much more complementary to the transition state than to substrates, so enzyme binds transition state much more tightly than it binds substrate -- free energy of TS (‡) is reduced but free energy of ES isn't much different from free energy of E + S
        Result: activation energy (DG) is dramatically decreased, so rate constant is increased (good catalysis!)

    • Fig. 8-6 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): Role of binding energy in catalysis
      • To lower activation energy for reaction (DG), system must acquire an amount of energy equivalent to the amount by which DG is lowered (DDG).
      • Much of this energy comes from the binding energy (DGB in figure below) contributed by formation of noncovalent interactions between "substrate" and enzyme in the transition state that are stronger than the interactions of enzyme with ground state of substrate.
      • The same types of interactions that provide catalytic power (binding S and especially binding TS () to lower DG) also provide specificity, the ability to discriminate among different potential substrates.
    • How much binding energy is needed to account for the large rate accelerations brought about by enzymes?
    • For a 106-fold increase in rate (a rate enhancement of 106 ), DDG = 34.3 kJ/mol.  This amounts to the formation of two additional hydrogen bonds between the enzyme and the transition state, as compared to the interaction between the enzyme and the substrate.

4. Induced fit:

    • Binding substrate to enzyme may stabilize a different conformation of either the enzyme or the substrate, orienting catalytic groups on the enzyme or promoting tighter transition state binding, and/or excluding water.
      • (This mechanism overlaps extensively with the mechanism above, that enzymes bind their transition states more tightly than their substrates.)
    • Fig. 8-21 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): Induced fit in hexokinase. Hexokinase Chime routine
      a)
      no substrate (glucose) bound, inactive conformation (active site residues not oriented properly to catalyze reaction)
      b) conformational change induced by glucose binding -- binding energy provided by binding of glucose (but no H2O) and the other substrate, Mg2+•ATP induces a conformational change to the active form (catalytic groups appropriately oriented relative to the substrates and each other to assist in the phosphoryl group transfer from ATP to glucose)

The rest of these catalytic mechanisms are more specific, involving properly positioned catalytic groups on the enzyme that aid in bond cleavage and formation.

5. General acid-base catalysis:

    • very often-used mechanism in enzyme reactions, e.g., hydrolysis of ester/ peptide bonds, phosphate group reactions, addition to carbonyl groups, etc.
    • Enzyme avoids unstable charged intermediates in reaction (which would have high free energies) by having groups appropriately located to
      • donate a proton (act as a general acid), or
      • accept a proton (abstract a proton, act as a general base)
    • If a group donates a proton (acts as a general acid) in chemical mechanism, it has to get a proton (a different one!) back (act as a general base) by end of catalytic cycle, and vice versa.
    • Protein functional groups that can function as general acid/base catalysts:
      His imidazole
      a-amino group
      a-carboxyl group
      thiol of Cys
      R group carboxyls of Glu, Asp
      e-amino group of Lys
      aromatic OH of Tyr
      guanidino group of Arg
      • Obviously, pH influences state of protonation of enzyme functional groups, so catalytic activity of enzymes using general acid-base catalysis is sensitive to pH.
    • Example: Mechanisms of uncatalyzed, acid-catalyzed, and base-catalyzed keto-enol tautomerization
      (redrawn from Fig. 11-6, Voet, Voet & Pratt, Fundamentals of Biochemistry, 1999)
      • General acid catalysis: partial proton transfer from an acid lowers the free energy of the high-free energy carbanionlike transition state of the keto-enol tautomerization.
      • Alternatively, the rate can be increased by partial proton abstraction by a base.
      • Concerted acid-base catalyzed reactions involve both processes occurring simultaneously.

6. Covalent catalysis (also sometimes called nucleophilic catalysis):

  • very often used in enzyme mechanisms
  • rate enhancement by the transient formation of a catalyst-substrate covalent bond
  • side chains of His, Cys, Asp, Lys and Ser can participate in covalent catalysis by acting as nucleophiles
  • coenzymes pyridoxal phosphate and thiamine pyrophosphate function mainly by covalent catalysis
  • Example of covalent catalysis coupled to general acid-base catalysis:
    Fig. 8-10 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): First step in chymotrypsin-catalyzed peptide bond hydrolysis -- general base catalyzed formation of covalent acyl-enzyme intermediate

7. Metal ion catalysis:

  • metal ions often used for one or more of the following:
    • binding substrates in the proper orientation
    • mediating oxidation-reduction reactions
    • electrostatically stabilizing or shielding negative charges (electrostatic catalysis)
  • Metalloenzymes contain tightly bound metal ions: (usually Fe+2, Fe+3, Cu+2, Zn+2, or Mn+2)
  • Metal-activated enzymes contain loosely bound metal ions: (usually Na+, K+, Mg+2, or Ca+2)
  • Some prosthetic groups are metalloorganic compounds, e.g. heme

More examples of transition state stabilization by enzymes:

  • racemization of the amino acid proline:

  • Best inhibitors are the planar transition state analogs, pyrrole-2-carboxylate and D-1-pyrroline-2-carboxylate, which bind far more tightly than proline.
  • The tetrahedral substrate analog is a poor inhibitor (binds very weakly).

  • Catalytic antibodies (see also Box 8-3, pp. 270-273 in Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000)
    • Antibodies raised against a transition state analog (a stable compound) are enzymatically active; antibodies against the substrate have no enzymatic activity.
    • These antibodies have a binding site complementary to the analog, that thus binds and stabilizes the transition state, and indeed they catalyze the reaction.
    • Catalytic antibodies are sometimes called "abzymes".
    • For reactions in which a "pentavalent" C (3 bonds and 2 "half bonds") is proposed in the transition state, the phosphorus analog can often be an excellent stable transition state analog, since P can have 5 bonds.
      • Example: antibodies against a transition state analog in ester hydrolysis (cyclic phosphate ester) catalyze the hydrolysis reaction:

Binding Energies of Enzymes and Substrates

  • Net binding energy represents the difference between the binding energy of substrate with water and the binding energy of substrate with enzyme.
  • Thus, Kd for an enzyme-substrate complex reflects the relative stabilities of substrate bound to the enzyme and substrate free in solution.
  • Kd depends on
    • strength of hydrogen bonding between E and S, compared with the combined strengths of their separate hydrogen bonding to water
    • stability of salt linkages between E and S, compared with tendency of individual ions to be solvated by water
    • Van der Waals (packing) interactions between S and E (not there when S is in water)
    • hydrophobic effect (an entropic contribution) that helps "drive" some substrates out of water into protein "interior" (binding site)
  • Although each difference might be small, when summed over all the interactions, the energy can be considerable. Note that hydrogen bonding and salt linkages between the enzyme and substrate are entropically favored due to release of water.

Utilization of enzyme-substrate binding energy in catalysis

  • Maximum binding energy between an enzyme and a substrate occurs when there's maximal complementarity between structure of binding site and structure of the substrate.
    • structure of "substrate" changes during reaction, as substrate becomes first the transition state and then products
    • structure of enzyme can only be maximally complementary to one form of the substrate
    • If structure of active site is complementary to the transition state, then binding increases as the reaction proceeds, which lowers the activation energy.
    • Having the active site bind the transition state most strongly also means that products are bound weakly, which favors dissociation of products from enzyme once reaction is complete.
    • When active site is complementary to transition state, the full "substrate" binding energy is only realized as the reaction reaches the transition state, which lowers activation energy of the reaction by the magnitude of the binding energy (DGB in Fig. 8-6 above).
  • Evolution/determination of the maximum rate of an enzyme-catalyzed reaction -- 2 components:
    1) strong binding of transition state, and
    2) weak binding of substrate

    • Rate of reaction per active site = v/[Et] = mols product formed per mol active site per sec
    • Maximizing Km at constant kcat/Km
    • Consider a hypothetical case where [S] = 10-3 M (1 mM, a "ballpark" concentration for a lot of metabolic intermediates) and kcat / Km = 106 M-1s-1.
    • To maintain the constant kcat/Km, kcat and KM must vary in tandem, as shown in the table below.
    • Rate per active site in table (calculated from equation above) demonstrates that rate of reaction per active site is maximal when [S] << Km (i.e. when Km >> [S]).

    Km (M)

    kcat (s-1)

    Rate per active site (s-1)

    10-6

    1

    1

    10-5

    10

    9.9

    10-4

    102

    100

    10-3

    103

    500

    10-2

    104

    909

    10-1

    105

    990

    1

    106

    999

    • This result contradicts the belief that tight binding of substrate, low Km, is an important component of enzyme catalysis, and is consistent with the principle that enzymes bind the TRANSITION STATE tightly.

  • The evolution of an enzyme to give maximal rate may be divided into two hypothetical steps
    • kcat/Km is maximized by having the enzyme be complementary to the transition state.
    • Concentration of free enzyme [E] is maximized by having Km be high relative to [S], which means that as much of the enzyme as possible is in the free state.
      • Remember, when [S] << Km, the Michaelis-Menten equation can be rewritten as v =[E][S] (kcat / Km)

  • HOWEVER, key regulated enzymes sometimes have a low Km.
    • A low Km (relative to [S] in the cell) may be advantageous for the first enzyme in a metabolic pathway, which generally is the rate-limiting step in the pathway, and there is not enough enzyme (or the enzyme is not active enough) for the reaction to operate at equilibrium in the cell -- the relative concentrations of substrates and products in vivo are far from equilibrium.
    • This step regulates entry of material into the pathway over a wide range of substrate concentrations -- the enzyme would always operate at maximal velocity (saturation with S), so [S] becomes irrelevant to the rate.
    • The rate can then be regulated only by altering the amount or the activity (kcat) of the enzyme -- these regulated enzymes are usually allosteric enzymes, whose activities are sensitive to metabolic signals in form of metabolites that serve as allosteric effectors.

Chemical mechanisms of specific enzymes that provide illustrations of one or many of the 7 general mechanisms above:

Induced fit

  • Hexokinase catalyzes ATP-dependent phosphorylation of glucose (transfer of phosphoryl group from ATP to glucose).
  • Binding of glucose to the enzyme induces a major conformational change - induced fit (hexokinase).

  • Phosphoglycerate kinase catalyzes phosphoryl transfer from 1,3-BPG to ADP, to form ATP.
  • The two substrates are brought together in the active site by a conformational change (pgk).

  • Kinases: enzymes that transfer phosphoryl groups between a nucleoside triphosphate (often ATP) and some other substrate.
  • To which of the 6 classes of enzymes do kinases belong?

  • An induced fit at an interface. Lipases are enzymes that hydrolyze esters of long chain fatty acids.  These enzymes show virtually no activity against water-soluble substrates and only hydrolyze substrates present in the interface between water and lipid - interfacial activation.  The activation of the lipases at the interface is a result of a conformational change, induced fit, caused by the enzyme binding to the interface.   This conformational change exposes the active site of the enzyme (lipase).
  • The Chime routine shows the structure of the enzyme in water, where the conformation has access to the active site serine blocked by a helix.
  • When lipase binds to the the interface created by its substrate, the helix moves, allowing the substrate to bind at the active site.

 

Serine proteases

  • Proteases catalyze hydrolysis of peptide bonds. (In vivo, function in digestion of nutrient protein, and in protein turnover (degradation of proteins that are old or no longer needed as conditions change).
    (To which of the 6 classes of enzymes do proteases belong?)
  • Equilibrium (in 55.5 M H2O) lies FAR to the right, but in the absence of a catalyst the reaction is extremely slow (fortunately -- or our bodies would all be puddles of amino acids in solution!)
  • Peptide bonds are thus "kinetically stable", or metastable.
  • Mechanism of uncatalyzed reaction is simple nucleophilic attack by the :O of the H2O on the carbonyl C of the peptide bond, forming a tetrahedral intermediate which then breaks down as the amine "half" of the original peptide leaves.
  • Reason uncatalyzed reaction is so slow is that partial double bond character of peptide bond makes its carbonyl carbon much less reactive than carbonyl carbons in, for example, carboxylate esters.
  • Catalytic task of proteases is to make that normally unreactive carbonyl group more susceptible to nucleophilic attack by H2O.

  • Serine proteases are endoproteases (enzymes that cleave internal peptide bonds in proteins). The serine protease mechanism illustrates not only proximity and orientation, but also:
    • Transition state stabilization.
    • Covalent catalysis, involving a catalytic triad of Asp, His and Ser in the active site.
    • general acid-base catalysis
    • electrostatic catalysis
  • Chymotrypsin enhances rate of peptide bond hydrolysis by a factor of at least 109

  • Fig. 8-18 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000) Chymotrypsin structure -- a) primary structure, b) tertiary structure (surface), R1 group (substrate) binding site in green, and active residues (including catalytic triad) in red.

    c) tertiary structure (ribbon, with active site residues), d) close-up of active site with portion of bound substrate shown. First (N-terminal) part of substrate is in green, with aromatic R1 group in blue. Active site Ser oxygen (red) attackes carbonyl C (last green atom in main chain; carbonyl O is purple). Peptide bond to be cleaved is between last green atom (carbonyl O) and 1st blue atom (amide N, coming out toward viewer). Amide nitrogens of oxyanion hole (transition state binding) are space-filling, in orange.

  • The enzyme-catalyzed mechanism doesn't involve direct attack on the carbonyl O by H2O -- the pathway is different.
  • Reaction occurs in 2 half-reactions
    • First half reaction("acylation phase") -- enzyme is acylated:
      • enzyme itself provides a potent nucleophile, a specific Ser OH group (which is made more nucleophilic than usual with the help of a nearby His residue that acts as a general base).
      • excellent example of nucleophilic catalysis: the enzyme itself attacks carbonyl C of scissile peptide bond, forming a covalent intermediate, the acyl-enzyme intermediate (which is actually a carboxylate ester of the carboxylate "half" of the original substrate, attached to the Ser R group as the alcohol component of the ester), and displacing first product, the C-terminal portion of the substrate, with its new N-terminal a-amino group. This "half" of the original peptide/protein is released as first product at the end of this first phase.
    • 2nd half reaction ("deacylation phase") -- enzyme is deacylated:
      • 2nd substrate, H2O, enters and acts as a nucleophile, attacking the carbonyl C of the the acyl enzyme (carboxylate ester), hydrolyzing the ester bond to displace second product, the N-terminal portion of the original substrate, with its new C-terminal a-carboxyl group, and regenerating the alcohol component (the enzyme with its Ser-OH free again); the carboxylic acid "half" of the original peptide/protein is released.
  • S = polypeptide; P1 = AMINE product (2nd "half" of substrate) R'-NH2
    E•P2 = COVALENT intermediate, the ACYL-ENZYME intermediate
    P2 = CARBOXYLATE product (1st "half" of substrate) R-COOH

  • Fig. 9.5 from Berg, Tymoczko & Stryer, Biochemistry, 5th ed., 2001): Covalent catalysis by chymotrypsin. (Chymotrypsin can catalyze hydrolysis of carboxylate esters as well as of amide bonds in peptides and proteins.)
  • What type of bisubstrate kinetic mechanism does this represent (sequential or ping-pong)?
  • All serine proteases have in the active site 3 amino acid residues known as the catalytic triad:
    Fig. 16.18 from Garrett & Grisham, Biochemistry, 2nd ed., 1999: Catalytic triad of chymotrypsin
  • The currently accepted serine protease mechanism follows below -- the same mechanism is used by other serine proteases, e.g. trypsin.
  • In the 2-phase process, the overall chemical steps in the 2nd phase are almost an exact repeat of the processes in the first phase.
  • Steps are numbered as in Fig. 8-19 in Lehninger Principles of Biochemistry, and it would be useful for you to study that figure as well, with its schematic indication of the active site and especially of the oxyanion hole, with its ONE hydrogen bond to the substrate, and TWO hydrogen bonds to the tetrahedral intermediates and presumably also to the transition states.
    • Color figures here, from Berg, Tymoczko & Stryer, have a couple of extra "steps" in mechanisms because they show dissociation of P1, binding of H2O, and dissociation of P2, all as separate steps, so ignore their numbering. (It's not the numbers of steps, it's what happens in what order that matters.)

CHEMICAL MECHANISM OF CHYMOTRYPSIN (color figures from Berg, Tymoczko & Stryer, Biochemistry, 5th ed., 2001, Fig. 9-8)
1. FORMATION OF ES COMPLEX

  • Enzyme binds substrate (a peptide or protein), with substrate's aromatic or bulky hydrophobic side chain ("specificity group"), R1, in a particular hydrophobic pocket ("specificity pocket") of the enzyme located sterically appropriately for cleavage of the peptide bond on carbonyl side (i.e., the "carboxyl" side) of that residue.
  • The polypeptide chain backbone of peptide substrate also forms a short b-sheet (hydrogen bonds) with the enzyme backbone in binding site (not shown in figures).
  • Carbonyl oxygen of substrate forms ONE hydrogen bond to an amide N-H in the enzyme backbone (not shown in these figures).
  • We now have an ES complex. What follows is a 2-phase process, with the overall chemical steps in the 2nd phase being almost an exact repeat of the processes in the first phase.

 

FIRST PHASE OF CATALYSIS (PHASE I, ACYLATION):
Formation of acyl-enzyme covalent intermediate and generation of P1, the amine product (In ester hydrolysis, P1 would be an alcohol.)
2. FORMATION OF FIRST TETRAHEDRAL INTERMEDIATE

  • Oxygen atom of active site Ser-OH is activated by hydrogen bond network linkage to His (imidazole ring N: ) in catalytic triad: His acts as a proton acceptor (general base catalysis), taking proton from Ser-OH to become HisH+, while the O atom becomes a potent nucleophile, to attack carbonyl C of peptide bond to be cleaved.
  • As His accepts the proton from Ser-OH (so His picks up a + charge), the Ser-O becomes a potent nucleophile and carries out a nucleophilic attack on carbonyl C of substrate (nucleophilic catalysis, i.e. covalent catalysis), forming a COVALENT bond to the carbonyl C. These 2 steps (loss of H+ by Ser-O, and attack of Ser-O on carbonyl C) are thought to be CONCERTED (occur at the same time), so Ser-O doesn't actually exist as an oxyanion (-O- ).
  • Asp in catalytic triad a) helps maintain perfect steric arrangement of His and Ser residues in hydrogen bonded network, and b) facilitates H+ transfer by electrostatic stabilization of HisH+ after it has accepted the proton.
  • Result of this step is the FIRST TETRAHEDRAL INTERMEDIATE whose structure presumably is similar to that of the TRANSITION STATES involved in its formation and breakdown, with a negatively charged "carbonyl" OXYGEN (not a carbonyl group anymore), an OXYANION.
  • TRANSITION STATE STABILIZATION: Active site binds oxyanion more tightly than it bound original carbonyl group of substrate. Another hydrogen bond (in addition to 1st hydrogen bond that formed with substrate carbonyl O) forms between tetrahedral oxyanion and another enzyme backbone N-H group (part of the "oxyanion hole" portion of active site).
    • 2nd hydrogen bond couldn't form to carbonyl form of oxygen (=O) because of structural change on forming the tetrahedral intermediate (and presumably the transition states) -- oxyanion of intermediate moves further into the oxyanion hole portion of active site than the carbonyl O; C-O single bond longer than C=O double bond.
    • Furthermore, the hydrogen bonds to negatively charged oxygen are stronger than to neutral O.
  • Fig. 9.9 from Berg, Tymoczko & Styer, Biochemistry, 5th ed., 2001: Oxyanion hole of chymotrypsin -- note the TWO hydrogen bonds between enzyme backbone N-H groups and the -O- (oxyanion) of the tetrahedral intermediate.

3. FORMATION OF ACYL-ENZYME INTERMEDIATE
The FIRST tetrahedral intermediate BREAKS DOWN:

  • the original amide (peptide) bond CLEAVES -- the HisH+ donates a proton to the amino "half" of the original substrate (HisH+ acts as a general acid), and
  • that amine product (RNH2) dissociates from the active site, i.e. P1 leaves.
  • The breaking of the amide bond (departure of the amine) results in conversion of the oxyanion back into a C=O, still covalently attached to the Ser residue of the enzyme, forming the ACYL-ENZYME INTERMEDIATE. Thus the original carbonyl group of the peptide bond is now a carbonyl group again, but it's covalently attached to the Ser-O, i.e. the acyl-enzyme has a covalent ESTER linkage made up of the "carboxyl half" of the original peptide substrate with the O from the Ser alcohol R group.

SECOND PHASE OF CATALYSIS (PHASE II, DEACYLATION):
Breakdown of acyl-enzyme covalent intermediate by reaction with water (HYDROLYSIS) and release of P2, the carboxylic acid product

4. BINDING OF THE SECOND SUBSTRATE, H2O, IN THE ACTIVE SITE
(Berg et al., color figure, number this step 5 -- they dissociated the amine P1 as their "step 4".)

5. FORMATION OF THE SECOND TETRAHEDRAL INTERMEDIATE

  • HOH forms a hydrogen bond with HisN: (just like Ser-OH did in the first phase) in a hydrogen bond network, and
  • His again acts as a general base, to become HisH+, activating the O from the H2O to make it a potent nucleophile, about to attack carbonyl C of acyl-enzyme intermediate (an ester).
  • As His accepts proton from HOH (so His picks up a + charge), the O of the water becomes a potent nucleophile and carries out a nucleophilic attack on carbonyl C of acyl-enzyme intermediate (nucleophilic catalysis, i.e. covalent catalysis), forming a COVALENT bond to carbonyl C.
  • Asp in catalytic triad a) helps maintain perfect steric arrangement of hydrogen bonded network, and b) facilitates H+ transfer by electrostatic stabilization of HisH+ after it has accepted the proton.
  • Result of this step is SECOND TETRAHEDRAL INTERMEDIATE whose structure presumably is similar to that of the TRANSITION STATES involved in its formation and breakdown, with a negatively charged "carbonyl" OXYGEN, an "OXYANION". [Note that this SECOND tetrahedral intermediate has an -OH group on it (from the water) instead of the amido group of the amine "half" of original substrate in the FIRST tetrahedral intermediate.]
  • TRANSITION STATE STABILIZATION: Active site binds the oxyanion of second tetrahedral intermediate more tightly than it bound the carbonyl group of the acyl-enzyme.
    • Again, a second hydrogen bond forms between tetrahedral oxyanion and enzyme backbone in "oxyanion hole", that couldn't form to carbonyl oxygen of acyl-enzyme because of structural change on forming the intermediate.
    • Furthermore, the hydrogen bonds to negatively charged oxygen are stronger than to neutral O.

6. BREAKDOWN OF SECOND TETRAHEDRAL INTERMEDIATE

  • The original ester bond (from acyl-enzyme) CLEAVES -- the HisH+ (general acid) donates its proton back to the Ser O (generating the alcohol product of the hydrolysis of acyl-enzyme), as the ester bond breaks to generate the carboxylic acid component (R'COOH) from the acyl-enzyme (originally the N-terminal portion of the peptide substrate).
  • The carboxylic acid product then dissociates from the active site, i.e. P2 leaves.

  • The enzyme molecule is now in its original state, with the His imidazole in its neutral form, the catalytic triad appropriately hydrogen-bonded, and the active site ready to bind another molecule of substrate and do it all again.
  • Knowing that the His residue in the catalytic triad has to start out the catalytic cycle by accepting a proton from the active site Ser, and assuming a "generic" pKa for that His residue in a protein, sketch a plot of velocity vs. pH for the chymotrypsin-catalyzed reaction in the pH range from 4 to 10.

Divergent Evolution of Enzymes - Substrate specificity of pancreatic serine proteases

  • The pancreatic serine proteases are homologous -- they evolved by gene duplication and divergent evolution from a common ancestral gene.
  • Overall tertiary structure and the catalytic triads have been conserved.
  • Fig. 9.12, Berg, Tymoczko & Stryer, Biochemistry, 5th ed., 2001: 3o structures of chymotrypsin (red) and trypsin (blue).
  • Chime routine about serine proteases (Serine Proteases).
    • All serine proteases follow same mechanism -- specificity determined by nature of specificity pocket.
    • Serine proteases all have same substrate, a polypeptide chain, but different enzymes cleave chain adjacent to different amino acids.
      • Catalytic mechanism remains the same; specificity changed by altering aa residues in specificity pocket.
      • Chime routine comparing specificity of binding of R1 group of chymotrypsin with R1 group of elastase (Serine_Proteases_Evolution)
      • In trypsin specificity pocket, enzyme prefers to bind basic residues (R1 specificity group on substrate positively charged).
      • What change might you expect to find in structure of the specificity pocket of trypsin as opposed to chymotrypsin?
  • Chloroketones: inhibitors of serine proteases; bind in active site and react with His 57 (Chloroketone).

Convergent Evolution of Enzymes - Chymotrypsin and Subtilisin.

  • Subtilisins are a group of serine proteases produced by bacteria.
  • Both chymotrypsins and subtilisins hydrolyze polypeptide chains adjacent to large hydrophobic amino acid side chains - they have the same specificity.
  • However, the subtilisins and chymotrypsins have no amino acid sequence similarity and their tertiary structures are quite different
    • Chymotrypsin composed of primarily b-sheets; active site lies between two distinct domains. 
    • Subtilisin is an a/b-structure without domains.

chymo2.GIF (17175 bytes)
subtilisin2.GIF (18001 bytes)
Chymotrypsin      
  Subtilisin

  • Despite the differences in tertiary structure, the active sites of the two enzymes have very similar features: 
    • The catalytic triad, an oxyanion hole, a specificity pocket (not shown) and a nonspecific binding region, are all common features of the active sites of these two enzymes.
chymo.GIF (9594 bytes)
subtilisin.GIF (9815 bytes)
    Chymotrypsin  
Subtilisin
                              

What is the rest of the protein for?

  • It is usually stated that "the rest of the protein" is needed to establish the tertiary structure of an enzyme, which leads to formation of the active site.  That this is an oversimplification is shown by experiments which have attempted convert chymotrypsin to trypsin.
    • Chymotrypsin and trypsin have a very similar tertiary structure and the active site is also quite similar.
    • At first glance, it seems that the major difference is in the specificity pocket.  In chymotrypsin the bottom of the specificity pocket is Ser 189, while it is Asp 189 in trypsin.
    • So, substituting Asp for Ser at position 189 would seem to be all that would be necessary to convert chymotrypsin to trypsin, right?
      • When the experiment was done, what was obtained was an enzyme with very little activity toward either chymotrypsin or trypsin substrates.
    • Although extensive substitutions have been made in chymotrypsin, no one has yet succeeded in converting chymotrypsin to trypsin, although enzymes with altered specificity have been produced.
    • Clearly, some of "the rest of the protein" is important both in determining specificity and catalytic efficiency.  As yet, we can't offer a fuller explanation.
  • Pancreatic serine proteases are synthesized and secreted as inactive precursor proteins ("zymogens") and activated in the lumen of the small intestine.
    • PROBLEM: Accidental premature activation INSIDE the pancreatic cell would cause autodigestion of the pancreas, and could "snowball", as some active protease molecules could go on generating more and more --> pancreatitis.
    • Solution: intracellular INHIBITOR proteins that bind very tightly to the active protease and keep it from running amok in the cell.
    • Example: Pancreas contains a small protein (6 kDa) that is a potent inhibitor of trypsin ("pancreatic trypsin inhibitor").
      The inhibitor is a naturally occurring transition state analog of a protein substrate.
    • (Trypsin Inhibitor)
      • Pancreatic Trypsin Inhibitor (PTI) = very small intracellular protein that binds VERY tightly to the active site of trypsin
      • Inhibitor is a substrate analog (a Lys side chain on inhibitor binds to the Asp side chain in trypsin's specificity pocket, and also the backbone of the inhibitor makes backbone hydrogen bonds with the enzyme backbone in an antiparallel b sheet).
      • Inhibitor structure doesn't change on binding to trypsin, but the peptide bond involving the Lys specificity residue is distorted in the inhibitor structure -- it's nonplanar.
      • Inhibitor fits snugly into the oxyanion hole, behaving more like a transition state analog, but it's not flexible in the binding site the way a good substrate would be.
      • Result: that peptide bond (same one that would be cleaved faster if the inhibitor WERE a good substrate) IS in fact cleaved, but VERY, VERY SLOWLY (half life of several months!)

      • Another example: a1-antiproteinase (formerly called "a1-antitrypsin")
        • made in liver and secreted into the bloodstream (it's a plasma protein)
        • protects tissues from digestion by an elastase that's secreted by neutrophils (white blood cells that engulf bacteria)
        • really an anti-elastase (binds/inhibits elastase much better than it blocks trypsin)
        • binds very tightly (nearly irreversibly) to active site

        • genetic variant that (in homozygous individuals) results in only ~15% of normal serum levels --> unrestrained elastase activity that destroys alveolar walls in the lungs by digesting elastic fibers and other connective tissue proteins

        • RESULT: EMPHYSEMA, inability to expel enough CO2 from the lungs
        • Cigarette smoke inactivates the inhibitor by causing oxidation of an essential Met residue of inhibitor to Met sulfoxide, and modified inhibitor can't bind to elastase.

        • "Take-home message": DON'T SMOKE, especially if you're homozygous, or even heterozygous, for the trait that causes lower circulating levels of the inhibitor to start with!

Zymogen activation - creating the transition state-binding site

  • What is the basis for the lack of enzymatic activity of a zymogen, such as trypsinogen? 
  • As this Chime routine shows, the zymogen has a fully formed catalytic triad and specificity pocket, but lacks a functional oxyanion hole, which stabilizes the transition state (Zymogen).  
  • This is yet another example of the critical importance of transition state stabilization as the primary factor explaining the catalytic activity of enzymes.

Triose phosphate isomerase (PDF of this discussion of TIM mechanism)

  • Structure of triose phosphate isomerase (sometimes called "TPI" also):
    • the prototypical "TIM barrel" structure, an interior parallel b-barrel surrounded by a-helical interconnections between b strands (a/b barrel).
    • homodimer, each subunit having an active site near carboxyl end of b barrel.
  • Reaction catalyzed: isomerization of triose phosphates -- it interconverts glyceraldehyde 3-phosphate (an aldose) and dihydroxyacetone phosphate (the corresponding ketose) in glycolysis and gluconeogenesis.
  • Isomerization can proceed in absence of any catalyst, through an unstable enediol intermediate, but uncatalyzed reaction is so slow that the aldose and ketose are both stable compounds in absence of a catalyst.
  • Catalyst must increase the concentration of the enediol, by
    • a) lowering the free energy of the enediol intermediate (stabilizing the intermediate and thus presumably also stabilizing the transition states for its formation and breakdown), and
    • b) promoting the proton exchange required for enediol formation and thus increasing rate.
  • DGo' = - 7.5 kJ/mol for reaction shown above, so equilibrium favors DHAP.
  • kcat/Km = 4 x 108 M-1•sec-1 in the glyceraldehyde 3-P --> DHAP direction.
  • However, reaction is reversible, and active site accommodates either sugar as a substrate -- the enzyme's chemical mechanism is reversible. (A catalyst enhances the forward and reverse reaction rates by the SAME FACTOR; DDG is the same in both directions.)
  • In vivo, reaction is not at equilibrium:
    • When glyceraldehyde 3-phosphate is being removed in glycolysis (glucose catabolism), DHAP is converted to glyceraldehyde 3-phosphate.
    • When DHAP is needed (being removed) for gluconeogenesis (glucose synthesis), glyceraldehyde 3-phosphate is converted to DHAP.
    • But TIM is so efficient (kcat/Km ~2.4 x 108 M-1•sec-1) that the equilibrium ratio of the aldose and ketose is maintained even though metabolic flux is occurring.
  • The triose phosphate isomerase mechanism illustrates (Tim)
    • Transition state stabilization
    • Acid-base catalysis
    • Electrostatic catalysis
  • concerted acid-base catalysis:
    • Active site includes:
      • a Glu residue (Glu165) thought to be the base (B- ) in mechanism below
      • a His residue (His95, thought to be the HA in the mechanism below), and
      • a Lys residue (Lys12), thought to electrostatically stabilize the negatively charged transition state (an enediolate).
    • Enzyme provides a "cage" in which the substrate is held, by means of a "lid" (loop of the enzyme) that closes down over the bound substrate to trap and protect enediol intermediate from reacting with water (see Chime routine (Tim)).
    • Catalytic groups perfectly positioned to carry out acid-base catalysis and also to stabilize the enediol.
      • Mutation of the Glu to an Asp, moving the catalytic COO- group just 0.1 nm further from the substrate, results in a 1000-fold reduction in catalytic rate.

       

  •  

     

     

  • Chemical mechanism is thought to proceed as follows:
    • Enz-B- = Glu165; Enz-(HA) = His95

  • Many electron transfer reactions involve transferring electrons from one protein to another.  Here is one example in which ferredoxin-NADP+ oxido-reductase (FNR), containing a FAD, oxidizes the FeS protein, ferredoxin (Fd).
  • For small molecules that are substrates for enzymes, electrostatic interactions are often critical.  Here is an example involving sulfite oxidase. 
  • Many enzymatic reactions are complex and involve more than one substrate.  Here is an example involving glutathione reductase. 

Enzyme Catalysis: Summary

  • Enzymes accelerate reactions using 
    • Proximity and orientation effects
    • Electrostatic catalysis
    • Preferential Transition State Binding
    • Induced fit
    • General acid/base catalysis
    • Covalent catalysis
    • Metal ion catalysis

  • Enzymes accelerate chemical reactions by lowering the activation energy,  DG.
  • Strong binding of the transition state and weak binding of the substrate leads to the maximum rate because all the binding energy is used to lower DG.
  • Any process that uses some of the binding energy for another purpose, e.g., a protein conformational change, will lower the maximum rate.
  • Under physiological conditions, the maximum rate is achieved by maximizing kcat/Km AND having Km greater than the physiological [S]. This means that most of the enzyme will be free to interact with a substrate molecule and that every encounter will lead to a reaction.

Appendix: Chemical mechanism of lysozyme (not in Lehninger, and not included in class this semester because of lack of class time, so not on exams -- strictly enrichment if you're interested!)

  • There's a detailed discussion in Garrett & Grisham, Biochemistry, 2nd edition, 1999, pp. 526-530; this used to be used as a textbook in 462a,b, so there are probably copies in the library.
  • Lysozyme catalyzes the hydrolysis of the b(1--> 4) linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in bacterial cell wall polysaccharides and also the b(1--> 4)-linked poly NAG in chitin.
    • 6 sugar residues of the polysaccharide substrate fit into the active site as shown in the figure below (structure of lysozyme and of its active site) -- cleavage is between the 4th and 5th residues from the right in active site diagram at bottom of figure.
    • 4th sugar residue (adjacent to bond to be cleaved) has to be distorted out of its preferred chair conformation to fit into active site, and the inference is that the distorted structure is closer to the structure of the transition state for hydrolysis -- enzyme uses some of the favorable binding energy from binding the rest of the substrate structure to raise the substrate closer to the transition state, stabilizing the transition state relative to the ES complex.
    • Fig. 16.35 from Garrett & Grisham, Biochemistry, 2nd ed., 1999: Structure and active site of lysozyme
  • Hydrolysis reaction is accelerated also by participation of Glu35 and Asp52.
    • Bell-shaped pH-activity profile for lysozyme (see Lehninger Principles end-of-chapter problem #17, p. 292) suggests involvement of ionizable groups with pKa values of about 5.9 and 4.5.
    • Glu35 is in a nonpolar environment and has a higher than expected pKa, and has to be protonated at start of reaction because it first must donate a proton to the oxygen of the leaving group from hydrolysis (an alcohol), and subsequently accepts a proton from H2O, the 2nd substrate (see figure below).
    • Asp52 has a lower pKa, 4.5, close to expected value for an R group carboxyl, and it must be unprotonated for activity because it's required to stabilize carbonium ion intermediate (+) formed when the alcohol leaves, until carbonium ion can react with H2O, completing hydrolysis reaction:
      Fig. 16.37 from Garrett & Grisham, Biochemistry, 2nd ed., 1999) Lysozyme mechanism
    • The lysozyme mechanism illustrates
      • Transition state stabilization
      • Acid-base catalysis
      • Electrostatic catalysis

      • Several residues in the protein participate in substrate binding.
      • The binding of NAM4 in the chair conformation is unfavorable.
      • But the binding of residue 4 in the half-chair conformation is favorable (preferential transition state binding).
      • Hydrolysis involves acid-base catalysis:
        • Glu35 serves as a proton donor to the oxygen of the leaving alcohol. 
        • The resulting carbonium ion (+) is stabilized by the ionized side chain of Asp52 (electrostatic catalysis) until it can react with water.
    • (lysozyme)

  • Many glycosidases utilize a covalent intermediate in their mechanism.  Here is a good example of transition state stabilization using a bacterial glycosidase.
  • Another excellent example that clearly shows how transition state stabilization works is found in the structure of this transferase.


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Biochemistry 462a
http://www.biochem.arizona.edu/classes/bioc462/462a/462a.html
Department of Biochemistry and Molecular Biophysics
The University of Arizona
zieglerm@u.arizona.edu 
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Last revision fall 2003