5.3 How Enzymes Work: Catalytic Mechanisms

How Enzymes Lower Activation Energy to Increase the Reaction rate

Enzymes increase reaction rate (lower ΔG‡) by:

→ Participating in the Rxn (directly or thru cofactors)

• general acid/base catalysis = adding or removing a proton from the substrate via AA side chains of the enzyme

• nucleophilic/covalent catalysis = forms covalent bond b/w substrate & enzyme as an intermediate

• metal ion catalysis = metal ions of metalloenzymes can accept protons, binding/orientation of substrate, or stabilize TS

→ Proximity & Orientation: enzymes active sites line up the substrates or the Nuc & electrophile

• results in entropy reduction

→ Desolvation: substrate binding to the enzyme removes water for rxn to occur (done BEFORE rxn to decrease the required energy for the rxn)

→ Stabilization of the Transition state: enzymes make it easier to achieve TS

• “Preferential binding”: enzyme increases rxn rate by binding the TS better than the substrate

Participating in the Reaction: pH Sensitivity of Enzymes in Catalysis

pH sensitivity in catalysis is related to:

• Acid/base reactions

ex. His needs specific pH to maintain its protonation state

• Nucleophilic reactions

ex. Cys is a better Nuc when deprotonated

• Denaturation: enzyme could denature bc of pH change

→ enzymes operate maximally (most effective) at different pH’s

Participating in the Reaction: Acid/Base catalysis

General acid/base catalysis = adding or removing a proton from the substrate via AA side chains of the enzyme

• proton transfer/donation to/from specific side chains in the enzyme’s active site is FASTER than transfer to/from solvent (H2O), THUS results in rate enhancement

• NOT the same as specific acid-base catalysis, where H+ or OH- is the catalyst

→ enzyme’s residue side chain acting as:

• general acid = donates H to substrate

• general base = takes/accepts H from substrate

note: His residue can act as an acid & base

process:

1. w/o catalysis: unstable (charged) & intermediate breaks down to from reactants

   1. lone pair on OH’s O attack amide bond @ carbonyl

   2. form protonated oxygen & negative oxygen to connect reactants

2. proton transfers to & from water or a weak general acid, then general base stabilizes the intermediate → accelerates the rxn

   1. potion on O+ transfers to general base

   2. general acid transfers H to N → form protonated N

   3. O- form carbonyl & N group is released

Participating in the Reaction: Acid/Base catalysis - ex. RNase

RNase: carries out hydrolytic rxn of RNA → breaks down RNA into pieces

• an acid/base catalyst (enzyme participates in rxn to increase rxn rate)

• Christians folding study

• His residue of RNase’s active site acts as a general acid & base

• uses H2O

• breaks RNA at the phosphate connecting sugars

Mechanism of RNase (KNOW)

step 1: His 12 acts as a general base & His 119 as a general acid to promote nucleophilic attack & bond cleavage

1. His 12 acts as a general base: takes c2’s H

2. His 119 acts as a general acid: donates H to c6’s oxygen of the sugar leaving

3. bottom sugar group is released with an OH group at c6

4. H2O acts as a Nuc: attacks phosphate attached to c3 of top sugar

step 2: His 12 acts as a general acid & His 119 as a vernal base to promote hydrolysis

1. His 12 donates H to c2’s oxygen

2. His 119 takes H from the H2O attacking the phosphate

result:

• His residues of RNases active site return to original protonation state

• RNase ALWAYS generates a 3’ phosphate group: top sugar group has an OH from water attached to the phosphate

• c2’s oxygen is pronated again

→ proximity & orientation aspect of the mechanism: His’s are aligned well, THUS H’s barely have to move

Participating in the Reaction: Covalent Catalysis

nucleophilic/covalent catalysis = forms covalent bond b/w substrate & enzyme as an intermediate

• enzyme participates directly in enzymatic rxn by forming a transient covalent bond b/w enzyme & substrate

  • Nuc in enzyme becomes covalently attached

  • alters the pathway of the rxn: intermediates ARENT the same as the uncatalyzed rxns

→ Nucleophiles:

• hydroxyl group: ROH → RO-

• sulfhydryl group: RSH → RS-

• amino group: RNH3+ → RNH2

• imidazole group: His+ → His (deprot. N)

→ Electrophiles:

• protons: H+

• metal ions: M^n+

• carbonyl carbon atom

  • often the target bc c of C=O is an electrophilic centre

• cationic imine (schiff base): RR’C=NH+

Participating in the Reaction: ex. Reaction Mech involving covalent & general acid-base catalysis

→ this rxn mech involves covalent & general acid-base catalysis

• enzymes participates directly in the rxn using its Asp 102, His 57, and Ser 195 side chains in the active site

• forms a tetrahedral intermediate from the substrate

mech: general base catalysis & nucleophilic attack to form tetrahedral intermediate

• Enzyme-substrate complex:

1. Asp 102’s O is h-bonded to His’s N1

2. His 57 acts as a general base: His 57’s N3 takes H from Sea 195’s OH group

3. Ser 195’s O- acts as a Nuc and attacks substrates carbonyl C

4. form tetrahedral intermediate in enzyme’s actie site

Participating in the Reaction: Metal Ion catalysis

→ metal ions can participate in catalysis in 3 primary ways:

1. binding/orientation of substrate (not rly part of catalytic process)

2. stabilization of TS

ex. metal ion ca stabilize (-) on Oxygen

3. serving as reduction/oxidation centres

ex. metal ion is ox-reduc rxns

Participating in the Reaction: Metal Ion catalysis - ex. Carbonic anhydrase

• carbonic anhydrase is a metalloenzyme (can participate directly in rxns to increase rxn rate)

  • contains Zn2+ in active site that binds to intermed in rxn pathway

    • 3 His residues coordinate the Zn2+ in active site → increase nucleophelicity of OH- attached to zinc

→ carbonic anhydrase is a lyase (addition rxn) that is important in RBC’s to convert carbonic acid → bicarbonate + H+

mechanism occurring in metalloenzymes active site:

Proximity & Orientation → Entropy Reduction

Proximity & Orientation: want things to be near each/other & in the right orinetation for reaction to proceed

• analogous to substrate binding

• when 2 reactive groups are spatially constrained (near e/o) → increased rxn rate

• when 2 groups are constrained in space & orientation → even greater increase in rxn rate

  • even further enhancement

→ in an enzymes active site: 2 substrates or the substrate & active site groups are bound close together + in the correct orientation

THUS, active site of enzyme ensures proximity & orientation that increases rxn rate

Proximity & Orientation: ex. PEP carboxykinase

PEP carboxykinase: enzyme containing Mn2+ & MG2+ in its active site

• metal ions in the active site stabilize the (-) charges on the triphosphate group in its rxn

→ PEPCK’s active site brings the ATP (or GTP) & oxygens pyruvate close together, so the pyruvate’s O can become phosphorylated in the 2nd step of the rxn → allows rxn to proceed rapidly

• assisted by metal ion cofactors (stabilize negative charges)

Desolvation & Induced Fit

Desolvation: substrate binding to the enzyme removes water for rxn to occur

• why: water forms a shell around many small molecules → prevents reactive groups from reacting

  • substrate bindings removes many of these waters

→ should remove water BEFORE rxn, bc If done at the same time as the rxn it requires MORE energu

How:

• Induced fit = enzymes often change shape upon substrate binding

  • brings catalytic group into orientation & can be used to exclude water from active site

Desolvation & Induced Fit: ex. Hexokinase

hexokinase:

• a phosphotransferase

→ induced fit of HK: HK’s 2 domains change shape when glucose & ATP bind → brings substrates into right geometry & prevents water from hydrolyzing phosphoanhydride (excludes water)

→ Substrates of HK:

• beta-D-glucose: C6 OH group is phosphorylated- its the reactive group

• beta-D-xylose: space where c6 should be allows water to be present, THUS if xylose is bound at HK’s active site water is present → leads to phosphorylation of water (hydrolysis)

  • result: water & ATP react to have hydrolysis of ATP

→ ONLY want rxn to proceed is glucose is bound at active site

→ HK Mechanism at active site:

• uses non-covalent catalysis, THUS substrates react directly w e/o

  • enzyme (HK) provides Asp residue as a general base

  • phosphate of ATP acts as electrophile (has enhanced electrophility)

  • glucoses c6 -OH group acts as Nuc: O attacks ATP’s phosphate

  • leaving group is ADP

  • reactive groups: C6-OH

1. glucose’s c6-OH groups O attacks 3rd Phosphate of ATP

2. phosphorylates glucose @ c6 + releases ADP

3. Asp in HK’s active site acts as a general base: pulls proton off c6-OH group as the rxn proceeds

   

note: solved with crystallography

Stabilization of the Transition State (TS)

Stabilization of the Transition state: enzymes make it easier to achieve TS

• “Preferential binding”: enzyme increases rxn rate by binding the TS better than the substrate

→ differences in activation energies for catalyzed vs uncatalyzed rxns (binding energy) is ENHANCED by strong interaction with the TS, NOT the substrate

reminder: Binding energy = diff in activation energies for catalyzed vs uncatalyzed rxns

ΔGB = ΔΔG‡cat