UCM Biochem Chapter 6

binding energy

free energy released in formation of a large number of weak interactions between the enzyme and substrate

what does binding energy do?

established substrate specificity and increases catalytic efficiency

induced fit

modification of shape of an active site in an enzyme that occurs upon substrate binding

strategies to catalyze specific reactions

covalent, general acid-base, catalysis by approximation, metal ion

covalent catalysis

active site contains a reactive group that becomes temporarily covalently modified

Example of covalent catalysis

chymotrypsin

general acid-base catalysis

a molecule other than water plays a role of a proton donor or acceptor

Examples of general acid-base catalysis

histidine residues in chymotrypsin, carbonic anhydrase

catalysis by approximation

increases in the rate of a reaction that occur by bringing multiple substrates together along a single binding surface of an enzyme

Examples of catalysis by approximation

carbonic anhydrase binds carbon dioxide and water in adjacent site

metal ion catalysis

metal acts as an electrophilic catalyst by stabilizing a negative charge on the reaction intermediate, generates a nucleophile by increasing the activity of nearby molecules, or increases the binding energy of a enzyme-substrate interaction by binding to substrates

Examples of metal ion catalysis

zinc (2) helps form hydroxide in carbonic anhydrase, magnesium (2) stabilizes negative charge on rxn intermediate in EcoRV

protease

• cleave proteins by hydrolysis rxn (digest protein) and add a water molecule to peptide bond

• chops protein into smaller things to be absorbed on carbonyl side

chemical nature of peptide bonds

• kinetically stable due to resonance structures

  • thus they have double bond character

electrophilic (zap together)

tendency of a molecule to attract or acquire electrons

nucleophilic (bomb)

donate electrons or react at relatively electron- poor sites

what enzyme cleaves peptide bonds selectively on the carboxyl-terminal side of a large hydrophobic AA (methionine or phenylalanine)?

• good examples of covalent catalysis

• serine 195

chymotrypsin

catalytic triad

• Ser 195 sits in the cleft on the surface of chymotrypsin

a constellation of three residues, found in many proteolytic enzymes, in which two of the residues convert the remaining residue, usually a serine or cysteine residue, into the potent nucleophile

What does trypsin S1 pocket have that attracts and stabilizes (+) charged arginine or lysine residue in the substrate?

• which helps with enzyme specificity

aspartate residue (Asp 189) at bottom of pocket

What does elastase S1 pocket have that close off the mouth of the pocket so only small side chains can enter (alanine and serine)?

bulkier valine residues (Val 190 and Val 216)

oxyanion hole

region on certain proteolytic enzxymes that stabilizes the oxyanion constituent of the tetrahedral intermediate of the rxn

cysteine proteases

activated by the histidine to play role as nucleophile that attacks peptide bond. sulfur works as better nucleophile than serine

aspartyl protease

pair of aspartic acid residues act together to allow a water molecule to attack peptide bond. One Asp (deprotonated form) activates the attacking water by poising it for deprotonation. The other (protonated form) polarizes the peptide carbonyl group so it is susceptible to attack

metalloprotease

metal ion that activates water molecule to act as nucleophile to attack peptide carbonyl group.

protease main job (steps)

1. activate water molecule or another nucleophile

2. polarize peptide carbonyl group

3. stabilize a tetrahedral intermediate

How is one way carbonic acid is formed?

carbon dioxide reacts with water to make bicarbonate ion and a proton thus forms H2CO3

What is carbonic anhydrase?

• that makes rxn much faster

enzyme that hydrates carbon dioxide to form bicarbonate HCO3-

What does carbonic anhydrease contain that aids in increasing chemical reactivity?

metal ions specifically zinc 2+

ligand

molecule or group that binds to receptor including metal ions

In carbonic anhydrase, zinc is typically bound to ligands, which is usually four or more molecules, such as?

three sites are nitrogen ligands from imidazole rings of three histidine residues and one water molecule

When pKa is lowered to 7 from the binding of zinc and carbonic anhydrase, zinc attacks (nucleophile) carbon dioxide better than water

1. zinc ion facilitates the release of the proton from the water molecule which generates hydroxide ion

2. CO2 substrate bind to enzyme’s active site and is positioned to react with hydroxide ion

3. hydroxide ion attacks the CO2 , converting it into bicarbonate ion, HCO3-

4. catalytic site is regenerated with the release of HCO3- and the binding of another water molecule

The rate of CO2 hydration by carbonic anhydrase increases with the concentration of what?

A buffer (1,2-Dimethlybenzimidazole)

proton shuttle

group that facilitates the transfer of protons in or out of the active site of an enzyme

Carbonic anhydrase 2 has evolved a proton shuttle to allow buffer components to participate in the rxn from the solution. What is the primary component of this shuttle?

histidine 64

Four characteristics of enzymes

1. biomolecules that catalyze chemical rxns

2. composed of proteins and RNA

3. many require coenzymes and/or metal ions

4. accelerate chemical rxns by providing an alternate pathway for rxn to occur

Important unique features of enzymes

1. tremendous catalytic power (a lot of product and quick)

2. specificity (specific products formed)

3. regulation (enzymatic activity is finely controlled → turned on and off when needed)

enzyme specificity

each enzyme catalyzes a specific rxn on sm. range of substrates

Explain enzyme active sites (Enzyme increase rate of rxn in three major ways:)

• lower Ea by stabilizing the transition state

• provide an alternate path for product formation

• reduce entropy by orienting the substrates appropriately for rxn to occur

• change environment

• binding of substrate releases H2O

transition state model

an enzyme’s active site is designed to bind to TSǂ stronger than either the product or the reactant. The active site promotes the formation of the TSǂ

Four methods of enzyme catalysis

1. covalent

2. general acid-base

3. metal ion

4. proximity and orientation (approximation)

covalent catalysis

• Enzymes can form a weak covalent bond to a substrate molecule, which can stabilize a reaction intermediate

• A water molecule or second substrate then attacks the covalent intermediate

• AA are usually used a nucleophiles

• Coenzymes are usually electrophiles

general acid-base catalysis

• Donation of a H+ in a reaction by the enzyme or substrate and not a free H+ in solution

• AA side chains, N-Terminus and C-Terminus may be used

• place base next to H2O to make good hydroxide

metal ion catalysis

• Electrophile - stabilizes/or induces a negative charge on a substrate or intermediate

• May alter the acidity of a functional group creating a strong nucleophile

• May aid in substrate binding

• Electron transfer reactions

• add metal ion to make more electrophilic (partial (+) or polar)

proximity and orientation

• Reactions occur when a fruitful collision between reactants happen

• Enzymes bring the reactive species in close contact (increase the chance of collision)

• Enzymes place reactive groups in the correct orientation

• make sure rxn groups are allined

specificity of serine protease

determined by a binding pocket for substrates side chain

• pocket wouldnt form without disulfide bonds

serine protease catalytic triad

• Ser 195 acts as nucleophile

• His 57 acts as a general acid/base

• Asp 102 stabilizes and positions

• His57 in correct orientation