Mechanisms of Enzyme Action

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• Mechanisms of Enzyme Action

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Transition (TS) State Intermediate

• Transition state unstable high-energy
  intermediate
• Rate of rxn depends on the frequency at which
  reactants collide and form the TS
• Reactants must be in the correct orientation and
  collide with sufficient energy to form TS
• Bonds are in the process of being formed and
  broken in TS
• Short lived (1014 to 10-13 secs)

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Transition State (TS) Intermediate

• Activation Energy (AE) The energy require to
  reach transition state from ground state.
• AE barrier must be exceeded for rxn to proceed.
• Lower AE barrier, the more stable the TS
• The higher TS, the move likely the rxn will
  proceed.

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Intermediates

• Intermediates are stable.
• In rxns w/ intermediates, 2 TSs are involved.
• The slowest step (rate determining) has the
  highest AE barrier.
• Formation of intermediate is the slowest step.

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Catalyst stabilize the Transition State

• Enzyme binding of substrates decrease activation
  energy by increasing the initial ground state
  (brings reactants into correct orientation)
• Need to stabilize TS to lower activation energy
  barrier.

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Common types of enzymatic mechanisms

• Substitutions rxns
• Bond cleavage rxns
• Redox rxns

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Substitution Rxns

• Nucleophillic Substitution
• Direct Substitution

Nucleophillic e- rich Electrophillic e- poor
transition state
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Cleavage Rxns

• Heterolytic vs homolytic cleavage
• Carbanion formation (retains both e-) R3-C-H
  ? R3-C- H
• Carbocation formation (lose both e-) R3-C-H ?
  R3-C H-
• Free radical formation (lose single
  e-) R1-O-O-R2 ? R1-O O-R2

Hydride ion
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Oxidation reduction (Redox) Rxns

• Loose e- oxidation (LEO)
• Gain e- reduction (GER)
• Central to energy production
• If something oxidized something must be reduced
  (reducing agent donates e- to oxidizing agent)
• Oxidations removal of hydrogen or addition of
  oxygen or removal of e-
• In biological systems reducing agent is usually a
  co-factor (NADH of NADPH)

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Polar AA Residues in Active Sites
Reactive Charge AA Group _at_pH
7 Functions Aspartate -COO- -1 Cation Binding,
H transfer Glutamate -COO- -1 Cation Binding, H
transfer Histidine Imidazole 0 H
transfer Cysteine -CH2SH 0 Binding of acyl
groups Tyrosine Phenol 0 H-Bonding to
ligands Lysine -NH3 1 Anion binding, H
transfer Arginine Guanidinium 1 Anion
binding Serine -CH2OH 0 Binding of acyl groups
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Acid-Base Catalysis

• Accelerates rxn by catalytic transfer of a proton
• Involves AA residues that can donate or transfer
  protons at neutral pH (e.g. histidine)
• HB B H

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Acid-Base Catalysis
carbanion intermediate
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Covalent Catalysis

• 20 of all enzymes employ covalent catalysis
• A-X B E lt-gt BX E A
• A group from a substrate binds covalently to
  enzyme (A-X E lt-gt A X-E)
• The intermediate enzyme substrate complex (A-X)
  then donates the group (X) to a second substrate
  (B) (B X-E lt-gt B-X E)

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Covalent Catalysis

• Protein Kinases
• ATP E Protein lt-gt ADP E Protein-P
• A-P-P-P(ATP) E-OH lt-gt A-P-P (ADP) E-O-PO4-
• E-O-PO4- Protein-OH lt-gt E Protein-O- PO4-

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Binding Modes of Enzymatic Catalysis

• Proximity Effects
• Transition State Stabilization

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Proximity Effects

• In multi-substrate reactions, collecting and
  correct positioning of substrates important
• Increases effective concentration of substrates
  which favors formation of TS
• Decreases activation energy by decreasing entropy
• Increases rate of reaction gt 10,000 fold
• E binding to S can not be too strong or
  conversion from ES to TS would require too much
  energy

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ES complex must not be too stable

• Raising the energy of ES will increase the
  catalyzed rate
• This is accomplished by loss of entropy due to
  formation of ES and destabilization of ES by
• strain
• distortion
• desolvation

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ES complex must not be too stable

• Raising the energy of ES will increase the
  catalyzed rate
• This is accomplished by loss of entropy due to
  formation of ES and destabilization of ES by
• strain
• distortion
• desolvation

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Transition State Stabilization

• Equilibrium between ES lt-gt TS, enzyme drives
  equilibrium towards TS
• Enzyme binds more tightly to TS than substrate

Transition state analog
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The Serine Proteases

• Trypsin, chymotrypsin, elastase, thrombin,
  subtilisin, plasmin, TPA
• All involve a serine in catalysis - thus the name
  
• Ser is part of a "catalytic triad" of Ser, His,
  Asp (show over head)
• Serine proteases are homologous, but locations of
  the three crucial residues differ somewhat
• Substrate specificity determined by binding
  pocket

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Serine Proteases are structurally Similar
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Substrate binding specificity