Lecture31 Enzymes

7/31/2019 Lecture31 Enzymes

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Enzyme (Basic Principle)

What is enzyme?

Enzymeis 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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Active Site

Small relative to the total volume ofthe enzyme.

Crystal Structure of DNA Ligase


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Active Site


Usually occur in clefts and crevices in

the protein. Excluding solvents whichwould otherwise reduce the catalyticactivity of the enzyme.


QuickTime and a

TIFF (Uncompressed) decompressorare needed to see this p icture.


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Active Site


Usually occur in clefts and crevices in

the protein. Excluding solvents whichwould otherwise reduce the catalyticactivity of the enzyme.

Amino acids and cofactors are held inprecise arrangement with respect tostructure of the substrate.

The specificity of substrate utilizationdepends on the defined arrangementof the atoms in the enzyme activesite (complements the structure ofthe substrate molecule).



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I. Rate Acceleration

Enzyme acceleratethe rate of reaction:

Enhance the rate of reaction bystabilizing the transition stateof

the reaction.

Enzyme catalysis do not alter theequilibrium of a reversiblereaction.

E + S --> ES --> [EX*] --> EP --> E + P


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II. Binding Energy in Catalysis:

In most case, initial interaction isnoncovalent (ES) making use ofhydrogen bonding, electrostatic,hyodrophobic and van der Waalsforce to effect binding.

ES: Catalytic groups are now anintegral part of the samemolecule, the reaction of enzymebound substrates will follow firstorder rather than second orderkinetics.

(weak)

E + S --> ES --> [EX*] --> EP --> E + P


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II. Binding Energy in Catalysis:

Change in free energy GB.Favorable interaction betweenthe enzyme and substrate resultin a favorable intrinsic bindingenergy.

Entropy is lost when substratebinds to the enzyme.

(a) Two entities become one.

(b) Substrate is less able to rotate.

(c) Substrate become more ordered.

Weak interactions between theenzyme and substrate areoptimize and stabilize thetransition state.

(weak)

E + S --> ES --> [EX*] --> EP --> E + P

(stronger)


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III. Other factors involved in rate acceleration.

Desolvation:

When substrate binds to the enzyme surrounding water in solution is replacedby the enzyme. This makes the substrate more reactive by destablizing thecharge on the substrate.

Expose a water charged group on the substrate for interaction with theenzyme.

Also lowers the entropy of the substrate (more ordered).



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III. Other factors involved in rate acceleration.

Strain and Distortion:

When substrate bind to the enzyme, it may induces a conformational changein the active site to fit to a transition state.

Frequently, in the transition state, the substrate and the enzyme have slightlydifferent structure (strain or distortion) and increase the reactivity of thesubstrate.


Rate: 108 1

cyclic phosphate ester Acylic phospodiester


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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 reactionintermediate.


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Enzyme serves as a template to bind the substrates so that they are close to eachother in the reaction center.

- Bring substrate into contact with catalytic groups or other substrates.

- Correct orientation for bond formation.

- Freeze translational and rotational motion.

Approximation


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a) Bimolecular reaction(highactivation energy, low rate).

b) Unimolecular reaction, rateenhanced by factor of 105 due toincreased probability ofcollision/reaction of the 2 groups

c) Constraint of structure to orientgroups better (elimination offreedom of rotation around bondsbetween reactive groups), rateenhanced by anotherfactor of 103,for 108 total rate enhancementover bimolecular reaction

Approximation



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Covalent catalysisThe principle advantage of using an active site residue instead of waterdirectly 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 thesingle reaction directly.


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Covalent catalysis

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.

The principle advantage of using an active site residue instead of waterdirectly 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 ofthe single reaction directly.


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Covalent catalysis

ATP-Dependent DNA Ligase

PhosphoramidateIntermediate

Lys N P O Nucleoside

O

O

H

H

+

O

P

O

OO

P

O

O

O

H2C

OH OH

OO

P

O

O

O

N

N N

N

H2N

NH2

H2C

OH OH

OO

P

NH

O

O

N

N N

N

H2N

LysLys

P

O

O

O

P

O

O

O+

ATP

Ligase Adenylate


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Covalent catalysisWhat 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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Covalent catalysisSchiff Base Formation

A Schiff base may form from the condensation of an amine with a carbonylcompound.

The Schiff base (protonated at neutral pH) acts as an electron sink thatgreatly stabilizes negative charge that develops on the adjacent carbon.

Stable Intermediate


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Covalent catalysisSchiff Base Formation

Enzymes that form Schiff base intermediates are typically irreversiblyinhibited by the addition of sodium borohydride (Na+ BH4

).

Borohydride reducesthe Schiff base and traps the intermediate such that itcan no longer be hydrolyzed to release the product from the enzyme.

This is often used as evidence for a mechanism involving an enzyme-linkedSchiff base intermediate.


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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 stateof the chemical reaction.

Protons from amino acid side chains, cofactors, organic substrates act asBronsted-Lowry acid and base group.


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Acid-base catalysis

Transition State of Stabilization by a General Acid (A) or General Base (B) in EsterHydrolysis by Water.

Transition state can be stabilized by

acid group (A-H) acting as a partialproton donor for carbonyl oxygen of theester -Enhance the stability of partialnegative charge on the ester.

Alternatively, enzyme can stabilizetransition state by basic group (B:)

acting as proton acceptor.

For even greater catalysis, enzyme canutilize acid and base simultaneously


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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 of3 nucleotide.

His 119

General acidDonates a proton to 5 hydroxyl ofnucleoside.


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Acid-base catalysis


Example: RNase A:

His 12

General Base


His 119


2-3 cyclic phosphate intermediate

Net Proton Transfer from His119 to His12


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Acid-base catalysis


Example: RNase A:

His 12

General Base


His 119


Water replaces the released nucleoside

Acid and base roles are reversed for H12 and H119


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Acid-base catalysis


Example: RNase A:

His 12

General Base


His 119

General acid

Donates a proton to 5 hydroxyl ofnucleoside.

Original Histidine protonation states are restored


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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 pHand 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 oxidationstate.


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Metal ions can

Electrostatically stabilizing or shielding negative charges.

Act much like a proton but can be present in high concentration at neutral pHand 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 oxidationstate.


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1) Can stabilize developing negative charge ona leaving group, making it a better leavinggroup.


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2) Can shield negative charges on substrategroup that will otherwise repel attack ofnucleophile.


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2) Can shield negative charges on substrategroup that will otherwise repaile attack ofnucleophile.

3) Can increase the rate of a hydrolysisreaction by forming a complex with water,thereby increasing waters acidity.


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Examples:

Lecture31 Enzymes

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