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Recommended problems from the end of chapter 14: 3,5,8,9,10,12,13,14,15,16,17,18

Extent of catalytic rate enhancement by enzyme catalystsAs originally postulated by Linus Pauling and later shown using Eyring transition state theory, the extent of reaction rate enhancement by enzymes is directly related to the strength of transition state binding:

As well, most of the catalytic power of enzymes comes from transition state stabilization, such that:

kcku=KSKT

kcatku

= e(ΔGu

∓−ΔGc∓

RT)

2

Catalytic mechanisms

Enzymes utilized their functional groups to align the substrate within the active site such thatcatalysis can occur. In the absence of this 3-dimensional alignment the rate of catalysis is predicted to be very slow. Enzymes can accelerate the rate of reactions 106 to 1012 fold.

The types of catalytic mechanisms that enzymes employ can be classified into several groups:

1. acid-base catalysis 2. covalent catalysis 3. metal ion catalysis 4. electrostatic effects 5. proximity and orientation effects 6. preferential binding of transition state complex 7. proton-electron tunneling

Proximity and orientation effects• To promote catalysis, enzyme must bring the substrate(s) into the correct orientation andproximity.• Typically, proximity effects alone are probably not sufficient to account for reaction rate enhancements by enzymes. In addition to bringing the reactive groups close in space, enzymes must orient the reactive groups in the proper arrangement for a productive reaction.• In addition, it seems that enzymes can “freeze” the relative translational and rotational motions of the substrate(s) to achieve optimal orientation for chemical activity.• The above strategies “cost” free energy to the system because the entropy of the substrates decreases when they are made to position themselves in a particular orientation. How do enzymes overcome this energetic “cost?”

productive unproductive

3

Preferential transition state binding

In addition to simply positioning the reactants such that a reaction is likely, an enzymemay bind the transition state preferentially and thereby stabilize it. This can be done in anumber of ways: 1. The enzyme may bind the transition state more tightly than the substrate(s) or product(s). 2. An enzyme may mechanically strain the substrate through preferential binding of distortedsubstrates. This may “push” the substrate towards the transition state faster than when theenzyme binds substrates without steric strain. Support for this notion is the observation thatin many cases enzymes bind poor substrates with greater affinity in comparison to “good" substrates, which are bound less tightly.

R

CH2OH

R

CH2OH

C

C

O

O

H H

+ H2O

steric s train

When R = CH3 then reactionproceeds 315 faster than whenR = H, due to steric repulsion between the methyl groups and the reacting groups.

Figure 14-13ac p460

Low barrier hydrogen bonds and tunneling between donor and acceptor

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Direct electron/hydrogen tunneling

If the wavelength of a particle is similar to the distance through which it must move during a reaction, then it is possible for this particle to be transferred without going through a high energy transition state.According to the de Broglie equation,

€

λ =h

2mE

Where h is Planck’s constant, m is the mass of the particle, and E is the kinetic energy of the particle. In biochemical systems, only electrons and hydrogen ions have wavelengths that would be consistent with their tunneling under a transition state barrier.

If the kinetic energy is 10 kJ/mol, the de Broglie equation yields 0.9 Å for hydrogen, consistent with a sigma bond length.

Electrostatic effects in enzymatic catalysis - altered pKa values at the active site

• If the active site excludes water, the local environment within the site resembles thatof a non-polar solvent. This results in two main consequences:

1. The electrostatic interactions become stronger because Coulombic interactions are inversely proportional to the dielectric constant of the solvent.

2. In comparison to aqueous solutions, the pKa values of uncharged protonated groups, e.g., carboxylates, become elevated due to the lower stability of charged groups in non-polar solvents. Similarly, the pKa values of charged protonated groups, e.g., amines, become lower in non-polar solvents.

These effects can be modulated by the proximity of charged groups to the protonated groups, so individual pKa values must be determined experimentally.

• The charge distribution around the active site can stabilize the transition state(s).

• In several enzymes, charge distribution may “guide” the substrate to the active site.

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Acid-base catalysisThis is a very common mechanism in enzymatic catalysis.GENERAL ACID catalysis is a process in which partial proton transfer from an acid lowers thefree energy of a transition state (∆G‡) in a reaction.GENERAL BASE catalysis is a process in which partial proton abstraction by a base lowers thefree energy of a transition state (∆G‡) in a reaction.Many enzymes exhibit both processes, thus are called general acid-base catalysts.

Keto-enoltautomerization

uncatalyzed

general acidcatalysis

general basecatalysis

Acid-base catalysis• General acid - general base catalysis is common in biological systems.

• Amino acid residues that can be used in such processes include aspartic acid, glutamic acid, histidine, cysteine, tyrosine, and lysine. These residues can be made to have pKR near physiological pH, so they can participate as proton donors and acceptors.

effect of pH on reaction rate

3 4 5 6 7 8 9 10 11 12

pH

rate

6

Acid-base catalysis - reaction of RNase AX-ray crystallography, mutational analysis, pH dependence of the reaction rate, and the isolation of a 2’,3’-cyclic nucleotide intermediate helped elucidate the RNase A reaction mechanism.

Step 1. General base catalysis by His-12 results in abstraction of a proton from the 2’OH. This is followed by the formation of the 2’-3’-cyclic intermediate, which is promoted by His-119. His-119 acts as a general acid by protonating the leaving group.

Acid-base catalysis - reaction of RNase A

Step 2. The 2’,3’-cyclic nucleotide is hydrolyzed. His-12 is acting as a general acid by donating a proton to the leaving group, whereas His-119 is acting as a general base, abstracting a proton from water, which acts as the nucleophile and attacks the phosphorous of the phosphate group. The result is the release of a nucleotide with a 3’ phosphate group.

What experiments would youdesign to test this mechanism?

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Covalent Catalysis

Covalent catalysis involves the stabilization of the transition state by a transient covalent bondbetween enzyme and substrate.This mechanism also is known as nucleophilic catalysis because the enzyme usually initiatesthe reaction by nucleophilic attack on the substrate, which serves as the electrophile.

uncatalyzed

catalyzed

Covalent Catalysis 1. A primary amine, acting as a nucleophile, attacks the carbonyl group carbon. This resultsin the formation of a Schiff base. 2. Electrons are withdrawn from the reaction center by the catalyst, and the Schiff base decomposes quickly. 3. The catalyst is eliminated from the covalent intermediate by a reversal of the first reaction.

The catalytic groupmust be able to serveas an efficientnucleophile anda leaving group.This requires afunctional groupwith an abilityto “shuffle” electronsaround. Thesegroups include theside chain amino group of lysine, imidazole group ofhistidine, thiol groupof cysteine, and hydroxylgroup of serine.

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Metal ion catalysis

Transition metal ions are used in about a third of the known enzymes. These metals includeFe2+, Fe3+, Cu2+, Zn2+, Mn2+, and Co2+. In addition, main group metal ions such as Na+,K+,Mg2+, and Ca2+ are bound by many other enzymes and are essential for activity.

Metal ions: 1. Orient substrates properly for the reaction. 2. Mediate oxidation-reduction reactions within the active site. 3. Stabilize or shield negative charges.Metal ions are efficient negative charge ‘absorbers” because they can be present at high concentrations within the active site and may have charges greater than +1.

Carbonic anhydrase is a zinc-containing enzyme that catalyzes the following reaction:

CO2 + H2O ↔ HCO3- + H+

Metal ion catalysis - carbonic anhydrase

From the x-ray crystal structure itcan be seen that the zinc ion is found at the bottom of a 15Å active sitecleft.Histidine residues 94, 96, and 119are conserved. These residues coordinate the zinc within theactive site. In addition, a watermolecule is bound by the zinc ionat the active site.The arrow points towards the opening of the active site cavity.The light blue ligand reflects a probable additional coordination site for the zinc ion.

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Im = imidazole

1. The zinc-polarized water is ionizedthrough another conserved histidine.

2. The resulting OH- nucleophile attacksthe carbon of CO2, and resolution of theintermediate yields HCO3-.

3. Another water molecule is bound andthen ionized to regenerate the active site.

Im Zn2+ O-

Im

Im H

O

C

O

Im Zn2+ O

Im

Im H

CO

O-

H2O

Im Zn2+ O-

Im

Im H

+ H+ + H O C

O

O-

Metal ion catalysis - carbonic anhydrase

The lysozyme mechanism

Lysozyme cleaves bacterial cell walls. It hydrolyzes the β(1→4) glycosidic linkage fromN-acetylmuramic acid to N-acetylglucosamine (the NAM-NAG linkage)Hen egg white lysozyme is very abundant and therefore easy to purify. It is the most widelystudied lysozyme.

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Lysozyme structureThe structure of hen egg white lysozyme was solved by David Phillips in 1965. It is a smallprotein, of 14.6 kDa, and 129 residues.

This is a computer-generated model ofHEW lysozyme, with the Cα backbone in blue.The protein shape is globular-ellipsoid, withdimensions of 30 x 30 x 45 Å.Two residues especially important for catalysis are aspartate-52 and glutamate-35, indicated in white.Note the cleft in the enzyme. This cleft is where the substrate is predicted to bind.

Lysozyme structure

• The structure of the enzyme with the trisaccharide (NAG)3 was solved using x-ray crystallography.

• Here a six-sugar ring is shown, with the three additional residues modeled into the structure.

• It was difficult to modelin the 4th residue (residue D). This is because the C6 and O6 sugar atoms were too close to the protein residues.

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Substrate structure within the lysozyme active site

• In the natural substrate every secondresidue is n-acetyl muramic acid.

• The lactyl side chain cannot beaccommodated in the C or E positionin the enzyme. The NAM residues must be in positions B, D, and F, and theN-acetyl-glucosamine (NAG) residuesmust be in the A, C, and E positions.

• Lysozyme can cleave the NAM-NAGβ(1→4) linkage, and this can be achievedbetween residues B and C and/orresidues D and E.Because (NAG)3 is bound at positionsC, D, and E, it was suggested that the most probable cleavage site is between positions D and E.

• Using a different oligosaccharide, how would you test the idea that lysozyme cuts between positions D and E?

Lysozyme substrate structure

Distortion of the 4th sugar residue (residue D) from a chair conformation to a half-chairconformation solves the problem of accommodating this sugar residue into the active siteof lysozyme.

Note that residues C1, C2, C5, and O5 are coplanar in the half-chair conformation.

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Catalytic mechanism of lysozymeLysozyme catalyzes the conversion of an acetal to a hemiacetal via hydrolysis of a carbocation (oxonium ion) transition state.

Catalytic mechanism of lysozymeThis mechanism requires 1. A functional group that can act as a general acid catalyst. 2. Stabilization of the oxonium ion transition state by maintaining the R and R’ groups co-planar with the H, C, and O atoms. 3. Evidence that water is used in the second step to donate a hydroxyl group to the transition state and thereby stabilize the product.

• The functional groups in the immediate vicinity of the scissile bond are aspartate 52 and glutamate 35.

• Asp-52 is surrounded by polar residues with which it forms hydrogen bonds. It is thereforepredicted to have the typical aqueous solvent pKa of 3.9. Because at physiological pH this residue would be unprotonated, it cannot participate in as a general acid catalyst under those conditions.

• Glu-35, in contrast, sits in a mostly non-polar environment with little hydrogen bonding potential. The pKa of glu-35 can be altered (raised) by its hydrophobic microenvironment, resulting in a protonated carboxylic acid site chain. If this is in fact the case, glu-35 can act as a general acid catalyst.

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Lysozyme activity in H218O

Do these data support the mechanism presented in the previous slide?

Proposed catalytic mechanism of lysozyme

1. Residue D is distorted within the active site of the enzyme. 2. Glutamate-35 donates a proton to the leaving group R1-OH (general acid catalysis). 3. Asp-52 stabilizes the oxonium ion transition state by charge-charge interactions (electrostatic catalysis). This oxonium ion primarily is resonance-stabilized, and some lysozymes do not have asp-52. The structure of the transition state must be planar, and the structure of the bound substrate resembles that of the transition state (preferential binding of the transition state). 4. Water is utilized in a reversal of the preceding step. Glu-35 abstracts a proton from water(general base catalysis), the OH- becomes the nucleophile and it attacks C1, to which a hydroxylgroup is added to complete the cycle.

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Serine proteases

Serine proteases are a group of digestive enzymes with an essential serine residue.The enzymes differ in the nature of their specificity of the substrate.

Chymotrypsin is specific for a bulky hydrophobic residue preceding the scissile bondTrypsin is specific for a positively charged residue preceding the scissile bondElastase is specific for a small neutral residue preceding the scissile bond.

All these serine proteases are inactivated by DIPF, which irreversibly inactivates the enzyme.

(activ e serine) CH2OH + F P O

O

O

CH(CH3)2

CH(CH3)2

Di isop ropyl phospho fl ourid ate (DIPF)

(activ e serine) C H2O P O

O

O

CH(CH3)2

CH(CH3)2

+ HF

Enzyme -DIP

Serine proteases: reaction of a conserved histidine with TPCK inactivates the enzyme

H3C S

O

O

N H

C H

CH2

C

O

CH2Cl

Tosyl-L-phenylalanine chromomethylketone

Histidine 57 essential for the activity of chymotrypsin

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Structure of serine proteases

All serine proteases contain a serine, a histidine, and an aspartate residues that are essential for activity.

X-ray crystal structure of chymotrypsin (white) in a complex with eglin C, a substrate (blue).The catalytic residues are his-57 (blue), asp-102 (red) and ser-195 (yellow).Note how close serine-195 and histidine-57 are to the substrate.

The catalytic triad of chymotrypsin, a serine proteases

• Aspartate 102, histidine-57, and serine 195 form the catalytic triad of chymotrypsin. Theseresidues are conserved in all serine proteases.• The first step involve his-57 acting as a general base to abstract a proton from the serine-195 hydroxyl group. This is followed by a nucleophilic attack by the serine O- on the carbonyl carbonof the scissile peptide bond. • The process is enhanced by a polarizing effect of asp-102 on his-57 (electrostatic catalysis). Overall, this results in a tetrahedral intermediate.

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The catalytic mechanism of serine proteases

• Proton donation by his-57 (general acid catalysis)results in decomposition of the tetrahedralintermediate.

• This results in the formation of an acyl-enzymeintermediate (covalent catalysis).

The catalytic mechanism of serine proteases

The acyl-enzyme intermediate starts decomposing as the new N-terminus amine group (R’-NH2) becomes the leaving group, dissociates form the enzyme, and exchanges with water.

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The catalytic mechanism of serine proteases

• The acyl-enzyme intermediate decomposesby a reversal of the previous steps.

• His-57 abstracts a proton from water (generalbase catalysis), and the resulting hydroxyl groupattacks the carbonyl carbon.

• This results in the formation of anothertetrahedral intermediate.

The catalytic mechanism of serine proteases

• His-57 donates a proton to the tetrahedral intermediate (general acid catalysis).

• The new C-terminus of the polypeptide chain becomes the leaving group, and dissociatesfrom the enzyme as a carboxyl group. The enzyme now is “reset” and is ready for another round of catalysis.

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Transition state stabilization in chymotrypsin

• The carbonyl carbon cannot bind within the oxyanion hole because it is held within the activein such a way that it cannot “reach” that binding pocket.• In contrast, the carbonyl oxygen of the tetrahedral intermediate, which is negatively charged(oxyanion), can be bound within the binding pocket because it forms hydrogen bonds with the backbone NH groups of glycine 193 and serine 195.• Serine proteases preferentially bind the tetrahedral intermediate, and thus achieve transitionstate stabilization.