Mechanism of EnzymesMechanism of Enzymes

Mechanismsthe molecular details of catalyzed reactions

Nucleophilic substitution Nucleophilic substitution reactionsreactions

• Nucleophilic species are electron rich, and electrophilic species are electron poor

• Types of nucleophilic substitution reactions include:

(1) Formation of a tetrahedral intermediate by nucleophilic substitution

(2) Direct displacement via a transition state

Two types of nucleophilic Two types of nucleophilic substitution reactionssubstitution reactions

• Direct displacement

Two types of nucleophilic Two types of nucleophilic substitution reactionssubstitution reactions

• Formation of a tetrahedral intermediate

Cleavage reactionsCleavage reactions

• Carbanion formation

• Carbocation formation

Cleavage reactionsCleavage reactions

• Free radical formation

Catalysts Stabilize Transition StatesCatalysts Stabilize Transition States

Energy diagram for a single-step reaction

• Intermediate occurs in the trough between the two transition states

• Rate determining step in the forward direction is formation of the first transition state

Energy diagram for Energy diagram for reaction with intermediatereaction with intermediate

Enzymes lower the activation Enzymes lower the activation energy of a reactionenergy of a reaction

(1) Substrate binding

• Enzymes properly position substrates for reaction(makes the formation of the transition state more frequent and lowers the energy of activation)

(2) Transition state binding

• Transition states are bound more tightly than substrates (this also lowers the activation energy)

Enzymatic catalysis of the Enzymatic catalysis of the reaction A+Breaction A+B A-B A-B

Chemical Modes of Enzymatic Chemical Modes of Enzymatic CatalysisCatalysis

A. Polar Amino Acid Residues in Active Sites

• Active-site cavity of an enzyme is lined with hydrophobic amino acids

• Polar, ionizable residues at the active site participate in the mechanism

• Anions and cations of certain amino acids are commonly involved in catalysis

Acid-Base CatalysisAcid-Base Catalysis

• Reaction acceleration is achieved by catalytic transfer of a proton

• A general base (B:) can act as a proton acceptor to remove protons from OH, NH, CH or other XH

• This produces a stronger nucleophilic reactant (X:-)

General base catalysis General base catalysis reactions (continued)reactions (continued)

• A general base (B:) can remove a proton from water and thereby generate the equivalent of

OH- in neutral solution

Proton donors can also Proton donors can also catalyze reactionscatalyze reactions

• A general acid (BH+) can donate protons

• A covalent bond may break more easily if one of its atoms is protonated (below)

Covalent CatalysisCovalent Catalysis

Step one: a glucosyl residue is transferred to enzyme

*Sucrose + Enz Glucosyl-Enz + Fructose

Step two: Glucose is donated to phosphate

Glucosyl-Enz + Pi Glucose 1-phosphate + Enz

*(Sucrose is composed of a glucose and a fructose)

Triose Phosphate Isomerase Triose Phosphate Isomerase (TPI)(TPI)

• TPI catalyzes a rapid aldehyde-ketone interconversion

Proposed mechanism for TPIProposed mechanism for TPI

• General acid-base catalysis mechanism (4 slides)

Proposed mechanism for TPIProposed mechanism for TPI

Proposed mechanism for TPIProposed mechanism for TPI

Proposed mechanism for TPIProposed mechanism for TPI

Energy diagram for the TPI reactionEnergy diagram for the TPI reaction

Binding Modes of Enzymatic CatalysisBinding Modes of Enzymatic Catalysis

• Proper binding of reactants in enzyme active sites provides substrate specificity and catalytic power

• Two catalytic modes based on binding properties can each increase reaction rates over 10,000-fold :

(1) Proximity effect - collecting and positioning substrate molecules in the active site

(2) Transition-state (TS) stabilization - transition states bind more tightly than substrates

Binding forces utilized for Binding forces utilized for catalysiscatalysis

1. Charge-charge interactions

2. Hydrogen bonds

3. Hydrophobic interactions

4. Van der Waals forces

The Proximity EffectThe Proximity Effect

• Correct positioning of two reacting groups (in model reactions or at enzyme active sites):

(1) Reduces their degrees of freedom

(2) Results in a large loss of entropy

(3) The relative enhanced concentration of substrates (“effective molarity”) predicts the rate acceleration expected due to this effect

Reactions of carboxylates Reactions of carboxylates with phenyl esterswith phenyl esters

Reactions of carboxylates Reactions of carboxylates with phenyl esterswith phenyl esters

Reactions of carboxylates Reactions of carboxylates with phenyl esterswith phenyl esters

Weak Binding of Substrates to Weak Binding of Substrates to EnzymesEnzymes

• Energy is required to reach the transition state from the ES complex

• Excessive ES stabilization would create a “thermodynamic pit” and mean little or no catalysis

• Most Km values (substrate dissociation constants) indicate weak binding to enzymes

Energy of substrate bindingEnergy of substrate binding• If an enzyme

binds the substrate too tightly (dashed profile), the activation barrier (2) could be similar to that of the uncatalyzed reaction (1)

Transition-State (TS) Transition-State (TS) StabilizationStabilization

• An increased interaction of the enzyme and substrate occurs in the transition-state (ES‡)

• The enzyme distorts the substrate, forcing it toward the transition state

• An enzyme must be complementary to the transition-state in shape and chemical character

• Enzymes may bind their transition states 1010 to 1015 times more tightly than their substrates

Transition-state (TS) analogsTransition-state (TS) analogs

• Transition-state analogs are stable compounds whose structures resemble unstable transition states

• 2-Phosphoglycolate, a TS analog for the enzyme triose phosphate isomerase

Induced FitInduced Fit

• Induced fit activates an enzyme by substrate-initiated conformation effect

• Induced fit is a substrate specificity effect, not a catalytic mode

• Hexokinase mechanism requires sugar-induced closure of the active site

Glucose + ATP Glucose 6-phosphate + ADP

Lysozyme Binds Lysozyme Binds an Ionic Intermediate Tightlyan Ionic Intermediate Tightly

• Lysozyme binds polysaccharide substrates (the sugar in subsite D of lysozyme is distorted into a half-chair conformation)

• Binding energy from the sugars in the other subsites provides the energy necessary to distort sugar D

• Lysozyme binds the distorted transition-state type structure strongly

Bacterial cell-wall polysaccharideBacterial cell-wall polysaccharide

• Lysozyme cleaves bacterial cell wall polysaccharides (a four residue portion of a bacterial cell wall with lysozyme cleavage point is shown below)

Conformations of N-acetylmuramic acid Conformations of N-acetylmuramic acid

(a) Chair conformation

(b) D-Site sugar residue is distorted into a higher energy half-chair conformation

Lysozyme reaction mechanismsLysozyme reaction mechanisms

1. Proximity effects

2. Acid-base catalysis

3. TS stabilization (or substrate distortion toward the transition state)

Mechanism of lysozymeMechanism of lysozyme

Properties of Serine ProteasesProperties of Serine Proteases

Zymogens Are Inactive Enzyme Precursors

• Digestive serine proteases including trypsin, chymotrypsin, and elastase are synthesized and stored in the pancreas as zymogens

• Storage of hydrolytic enzymes as zymogens prevents damage to cell proteins

• Zymogens are activated by selective proteolysis

Activation of some Activation of some pancreatic zymogenspancreatic zymogens

Substrate Specificity of Serine Substrate Specificity of Serine ProteasesProteases

• Many digestive proteases share similarities in 1o,2o and 3o structure

• Chymotrypsin, trypsin and elastase have a similar backbone structure

• Active site substrate specificities differ due to relatively small differences in specificity pockets

Binding sites of chymotrypsin, Binding sites of chymotrypsin, trypsin, and elastasetrypsin, and elastase

• Substrate specificities are due to relatively small structural differences in active-site binding cavities

Identification of His at active siteIdentification of His at active site

• The irreversible inhibitor (TosPheCH2Cl) binds to the active-site His residue in serine proteases

Catalytic triadCatalytic triad of chymotrypsin of chymotrypsin

• Imidazole ring (His-57) removes H from Ser-195 hydroxyl to make it a strong nucleophile (-CH2O-)

• Buried carboxylate (Asp-102) stabilizes the positively-charged His-57 to facilitate serine ionization

-Chymotrypsin mechanism-Chymotrypsin mechanism

(Acyl E + H(Acyl E + H22O)O)

(E-TI(E-TI22))

(E-P(E-P22))

(E + P(E + P22))