Enzyme Basic Principle

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Enzyme (Basic Principle)

• What is enzyme?
• Enzyme is protein or nucleic acid.
• Chemical reactions
• breaking, forming and rearranging bonds.
• Specificity
• Dictated by the enzyme active site.
• Some active sites allow for multiple substrates.
• Cofactors
• Amino acid side chains have a limited chemical
  repertoire.
• Vitamin derivatives, metals (minerals) can bind
  as co-substrates or remain attached through
  multiple catalytic cycles

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Enzyme (Basic Principle)

• Active Site
• Small relative to the total volume of the enzyme.

Crystal Structure of DNA Ligase
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Enzyme (Basic Principle)

• Active Site
• Small relative to the total volume of the enzyme.
• Usually occur in clefts and crevices in the
  protein. Excluding solvents which would otherwise
  reduce the catalytic activity of the enzyme.

Crystal Structure of DNA Ligase
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Enzyme (Basic Principle)

• Active Site
• Small relative to the total volume of the enzyme.
• Usually occur in clefts and crevices in the
  protein. Excluding solvents which would otherwise
  reduce the catalytic activity of the enzyme.
• Amino acids and cofactors are held in precise
  arrangement with respect to structure of the
  substrate.
• The specificity of substrate utilization depends
  on the defined arrangement of the atoms in the
  enzyme active site (complements the structure of
  the substrate molecule).

Crystal Structure of DNA Ligase
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Enzyme (Basic Principle)

• I. Rate Acceleration
• Enzyme accelerate the rate of reaction
• Enhance the rate of reaction by stabilizing the
  transition state of the reaction.
• Enzyme catalysis do not alter the equilibrium of
  a reversible reaction.

E S --gt ES --gt EX --gt EP --gt E P
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Enzyme (Basic Principle)
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Enzyme (Basic Principle)

• II. Binding Energy in Catalysis
• In most case, initial interaction is noncovalent
  (ES) making use of hydrogen bonding,
  electrostatic, hyodrophobic and van der Waals
  force to effect binding.
• ES Catalytic groups are now an integral part of
  the same molecule, the reaction of enzyme bound
  substrates will follow first order rather than
  second order kinetics.

E S --gt ES --gt EX --gt EP --gt E P
(weak)
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Enzyme (Basic Principle)

• II. Binding Energy in Catalysis
• Change in free energy ?GB. Favorable interaction
  between the enzyme and substrate result in a
  favorable intrinsic binding energy.
• Entropy is lost when substrate binds to the
  enzyme.
• Two entities become one.
• Substrate is less able to rotate.
• Substrate become more ordered.
• Weak interactions between the enzyme and
  substrate are optimize and stabilize the
  transition state.

E S --gt ES --gt EX --gt EP --gt E P
(weak)
(stronger)
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Enzyme (Basic Principle)

• III. Other factors involved in rate acceleration.
• Desolvation
• When substrate binds to the enzyme surrounding
  water in solution is replaced by the enzyme. This
  makes the substrate more reactive by destablizing
  the charge on the substrate.
• Expose a water charged group on the substrate
  for interaction with the enzyme.
• Also lowers the entropy of the substrate (more
  ordered).

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Enzyme (Basic Principle)

• III. Other factors involved in rate acceleration.
• Strain and Distortion
• When substrate bind to the enzyme, it may induces
  a conformational change in the active site to fit
  to a transition state.
• Frequently, in the transition state, the
  substrate and the enzyme have slightly different
  structure (strain or distortion) and increase the
  reactivity of the substrate.

cyclic phosphate ester
Acylic phospodiester
Rate
108
1
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Catalytic Strategies

• Catalysis by approximation
• In reactions that include two substrates, the
  rate is enhanced by bringing the two substrates
  together in a proper oirentation.
• Covalent catalysis
• The active site contains a reactive group,
  usually a powerful nucleophile that become
  temporarily covalently modified in the course of
  catalysis.
• General acid-base catalysis
• A molecule other than water plays the role of a
  proton donor or acceptor.
• Metal ion catalysis
• Metal ions can serve as electrophilic catalyst,
  stabilizing negative charge on a reaction
  intermediate.

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Catalytic Strategies

• Approximation

Enzyme serves as a template to bind the
substrates so that they are close to each other
in the reaction center. - Bring substrate into
contact with catalytic groups or other
substrates. - Correct orientation for bond
formation. - Freeze translational and
rotational motion.
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Catalytic Strategies

• Approximation

• Bimolecular reaction (high activation energy, low
  rate).
• Unimolecular reaction, rate enhanced by factor of
  105 due to increased probability of
  collision/reaction of the 2 groups
• 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

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Catalytic Strategies

• Covalent catalysis

The principle advantage of using an active site
residue instead of water directly is that
formation of covalent linkage leads to
unimolecular reaction, which is entropically
favored over the bimolecular reaction. Enzyme
that utilize covalent catalysis are generally two
step process formation and breakdown of covalent
intermediate rather than catalysis of the single
reaction directly.
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Catalytic Strategies

• Covalent catalysis

The principle advantage of using an active site
residue instead of water directly is that
formation of covalent linkage leads to
unimolecular reaction, which is entropically
favored over the bimolecular reaction. Enzyme
that utilize covalent catalysis are generally two
step process formation and breakdown of covalent
intermediate rather than catalysis of the single
reaction directly.

• Y should be a better leaving group than X.
• X is a better attacking group then Z.
• Covalent intermediate should be more reactive
  than substrate.

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Catalytic Strategies

• Covalent catalysis

ATP-Dependent DNA Ligase
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Catalytic Strategies

• Covalent catalysis
• What kind of groups in proteins are good
  nucleophiles

• Aspartate caboxylates
• Glutamates caboxylates
• Cystine thiol-
• Serine hydroxyl-
• Tyrosine hydroxyl-
• Lysine amino-
• Histadine imidazolyl-

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Catalytic Strategies

• Covalent catalysis
• Schiff Base Formation
• A Schiff base may form from the condensation of
  an amine with a carbonyl compound.
• The Schiff base (protonated at neutral pH) acts
  as an electron sink that greatly stabilizes
  negative charge that develops on the adjacent
  carbon.

Stable Intermediate
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Catalytic Strategies

• Covalent catalysis
• Schiff Base Formation
• Enzymes that form Schiff base intermediates are
  typically irreversibly inhibited by the addition
  of sodium borohydride (Na BH4).
• Borohydride reduces the Schiff base and traps
  the intermediate such that it can no longer be
  hydrolyzed to release the product from the
  enzyme.
• This is often used as evidence for a mechanism
  involving an enzyme-linked Schiff base
  intermediate.

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Catalytic Strategies

• Acid-base catalysis
• A proton (H) is transferred in the transition
  state.
• Specific acid-base catalysis
• Protons from hydronium ion (H3O) and hydroxide
  ions (OH-) act directly
• as the acid and base group.
• General acid-base catalysis
• Catalytic group participates in protein transfer
  stabilize the transition state of the chemical
  reaction.
• Protons from amino acid side chains, cofactors,
  organic substrates act as Bronsted-Lowry acid and
  base group.

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Catalytic Strategies

• Acid-base catalysis
• Transition State of Stabilization by a General
  Acid (A) or General Base (B) in Ester Hydrolysis
  by Water.

Transition state can be stabilized by acid group
(A-H) acting as a partial proton donor for
carbonyl oxygen of the ester - Enhance the
stability of partial negative charge on the
ester. Alternatively, enzyme can stabilize
transition state by basic group (B) acting as
proton acceptor. For even greater catalysis,
enzyme can utilize acid and base simultaneously
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Catalytic Strategies

• Acid-base catalysis
• Histidine pKa is around 7. It is the most
  effective general acid or base.
• Example RNase A

• His 12
• General Base
• Abstracts a proton from 2 hydroxyl of 3
  nucleotide.
• His 119
• General acid
• Donates a proton to 5 hydroxyl of nucleoside.

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Catalytic Strategies

• Acid-base catalysis
• Histidine pKa is around 7. It is the most
  effective general acid or base.
• Example RNase A

• His 12
• General Base
• Abstracts a proton from 2 hydroxyl of 3
  nucleotide.
• His 119
• General acid
• Donates a proton to 5 hydroxyl of nucleoside.

2-3 cyclic phosphate intermediate Net Proton
Transfer from His119 to His12
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Catalytic Strategies

• Acid-base catalysis
• Histidine pKa is around 7. It is the most
  effective general acid or base.
• Example RNase A

• His 12
• General Base
• Abstracts a proton from 2 hydroxyl of 3
  nucleotide.
• His 119
• General acid
• Donates a proton to 5 hydroxyl of nucleoside.

Water replaces the released nucleoside Acid and
base roles are reversed for H12 and H119
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Catalytic Strategies

• Acid-base catalysis
• Histidine pKa is around 7. It is the most
  effective general acid or base.
• Example RNase A

• His 12
• General Base
• Abstracts a proton from 2 hydroxyl of 3
  nucleotide.
• His 119
• General acid
• Donates a proton to 5 hydroxyl of nucleoside.

Original Histidine protonation states are restored
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Catalytic Strategies

• Metal ion catalysis.

• Metal ions can
• Electrostatically stabilizing or shielding
  negative charges.
• Act much like a proton but can be present in high
  concentration at neutral pH and can have multiple
  positive charges
• Act to bridge a substrate and nucleophilic group.
• Bind to substrates to insure proper orientation.
• Participate in oxidation/reduction mechanisms
  through change of oxidation state.

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Catalytic Strategies

• Metal ion catalysis.

• Metal ions can
• Electrostatically stabilizing or shielding
  negative charges.
• Act much like a proton but can be present in high
  concentration at neutral pH and can have multiple
  positive charges
• Act to bridge a substrate and nucleophilic group.
• Bind to substrates to insure proper orientation.
• Participate in oxidation/reduction mechanisms
  through change of oxidation state.

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Catalytic Strategies

• Metal ion catalysis.

• Can stabilize developing negative charge on a
  leaving group, making it a better leaving group.

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Catalytic Strategies

• Metal ion catalysis.

• Can stabilize developing negative charge on a
  leaving group, making it a better leaving group.
• Can shield negative charges on substrate group
  that will otherwise repel attack of nucleophile.

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Catalytic Strategies

• Metal ion catalysis.

• Can stabilize developing negative charge on a
  leaving group, making it a better leaving group.
• Can shield negative charges on substrate group
  that will otherwise repaile attack of
  nucleophile.
• Can increase the rate of a hydrolysis reaction by
  forming a complex with water, thereby increasing
  waters acidity.

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Catalytic Strategies
Metal ion catalysis. Examples