Chapter 14

Question 1

role of enzymes

Answer 1

favoring formation or stabilization of the transition state

• enzyme binds transition state more favorably then substrate
• energy of EX++ lowered more than energy of ES
• induced fit of ES complex helps to bring S to transition state
• “destabilize” ES, bringing it closer in energy to EX++

Question 2

Destabilizing ES complex

Answer 2

• entropy loss in ES formation
• Strain
• desolvation
• electrostatic effects

Question 3

Stabilizing EX++

Answer 3

• covalent catalysis
• general acid or base cat
• metal ion cat
• proximity and orientation (same as entropy loss in ES formation)

Question 4

loss of entropy in ES formation

Answer 4

entropy (amount of disorder) decreases upon binding of substrate to the enzyme
S alone free to undergo transitional motion

Question 5

Desolvation of solvent molecules

Answer 5

ex: removal of water on substrate to enzyme
- raises energy of the ES complex
more likely for the substrate to react at a faster rate bc barrier will be smaller

Question 6

Electrostatic “mismatches”

Answer 6

repulsion by charges causes the destabilization. enzyme raises the delta G of destabilization so the difference between them is small

Question 7

destabilizing ES

Answer 7

means you raise the energy of the ES complex and this will help cat the rxn

Question 8

another ground state destabilization: near attack conformations (NACS)

Answer 8

ground state conformers that readily convert to the transition state
- changes conformation of the substrate so that is ready for the reaction

Question 9

covalent cat

Answer 9

X-E, X is the nucleophile
- covalent bond formed with enzyme. formation of the covalent intermediate stabilizes the EX++ complex
- ping-pong mechanism
BX + Y -> BY + X
acceptor group on enzyme is better nuc than Y and Y is a better leaving group than X

Question 10

General Acid or Base cat.

Answer 10

• specific - hydrolysis by water (give H+ or OH-)

- general acid/base hydrolysis, acids or bases other than H+ or OH- cat the rxn (ex: aa)

Question 11

specific A/B hydrolysis

Answer 11

only pH matters w/ H+ or OH- diffuses into the active site from solvent

Question 12

General acid-base cat

Answer 12

both pH and buffer concentration affect rxn rate constant in transition state it can donate/accept H+

Question 13

ex of general acid-base rxn

Answer 13

catalysis of p-nitrophenylacetate hydrolysis by imidazole

Question 14

metal ion cat.

important in RNA

Answer 14

two types electro phallic cat

and providing a nucleophile

Question 15

electrophilic cat

Answer 15

stabilize increasing e- density or negative charge: stabilize the transition state

Question 16

providing nuc. metal ion cat

Answer 16

increasing acidity of a group with an ionizable H+

- in some ribozymes

Question 17

metal ions in cat by nucleolytic ribozymes

Answer 17

activate nuc.

• groups that could facilitate abstraction of protons
• stabilization of transition state
• stabilization of leaving group by metal ions
• conformational change to bring about nuc attack
• result is the product

Question 18

roles of metal ions

Answer 18

• general base (bronsted) cat - removal of proton from 2’OH
• inner sphere association w/2’oxygen
• stabilization of charge separation in transition state
• stabilization/protonation of oxyanion LG

Question 19

proximity effect

Answer 19

brings it closer to the functional groups that need to work together to get closer to the transition state so it happens quicker.
so when 2 functional groups are together on the same molecule they can react faster (entropy decreases?)

Question 20

orientation effect

Answer 20

steric crowding - help orient the functional group so that the rate of concentration can occur quicker so that the groups are closer together to react. with steric hindrance there is little mobility.

Question 21

Ktx

Answer 21

dissociation constant of the ES complex in the TS. affinity of the enzyme for the transition state

Question 22

transition state analogues

Answer 22

enzyme has a high affinity for the transition state, ktx. so if we have a structure that looks like the transition state than it can be an excellent inhibitor and can tell us a lot about the transition state since it occurs to quickly to isolate.
- racemization of proline
- deaminase of aldose and adenosine
hairpin ribozyme

Question 23

Statins

Answer 23

transition state analogue inhibitors of a key enzyme in biosyn of cholesterol and thus is a potent cholesterol lowering drug

Question 24

Km/K1

Answer 24

they are opposites Km affinity for transition state

K1 is affinity for the analogue of transition state

Question 25

transition state stabilization by a catalytic RNA

Answer 25

Vanadate (VO5) (inhibit the function of the ribozyme - so it's an analogue) stimulates the transition state of the phosphodiester bond - binds more tightly to the active site than the substrate or product - binding vanadate to ribozyme thus assisted in identification of the transition state

Question 26

examples of mechanisms of stabilization

Answer 26

- serine proteases | - aspartic proteases

Question 27

serine proteases - catalytic triad

Answer 27

his-57 asp-102 ser-195 found in active site

Question 28

serine protease

Answer 28

mixture of covalent and general acid-base cat

Question 29

protease

Answer 29

an enzyme that breaks down proteins and peptides.

Question 30

hydrophobic pocket - serine protease

Answer 30

- gives preference to bulky, hydrophobic side chain (chymotrypsin cleave aromatic rings) - stabilize the hydrophobic rings in aa found in active site

Question 31

serine protease | stabilization of transition state

Answer 31

- covalent bond formation turns trigonal C into tetrahedral C tetrahedral oxyanion intermediate stabilized by amide of gly-193 and ser-195

Question 32

serine protease: burst kinetics

Answer 32

indicates formation of intermediate

Question 33

serine protease: LBHB

Answer 33

H that has come close enough between two e-negative atoms to lower the energy of a h-bond. that energy used to move the reaction forward

Question 34

serine protease

Answer 34

bind substrate asp-102 orients his-57 - his-57 act like general base and w/draws a H to form ES complex - LBHB between Asp-102 and his-57 (this is the tetrahedral (covalent) intermediate) - proton donation by His57 makes a good leaving group NH2-R (1st product) - bond cleaves and O no longer has a charge (acyl intermediate) transition state analogues exist - H2O activated by imidazole N of His-57 removes H+ so OH- can attack ester bond of the enzyme's acyl intermediate - nuc attack by h2o - another tetrahedral intermediate forms (LBHB also forms) with the enzyme, and decays freeing enzyme - 2nd product released (carboxyl part with aromatic ring)

Question 35

serine protease: oxyanion hole

Answer 35

additional stabilization by h-bonds between c=o and the backbone of s195 and g-193 - stabilizes negative charge on oxygen on chymotrypsin in its covalent intermediate

Question 36

serine protease: inhibitor that binds to serine covalently so the entire enzyme is inactive

Answer 36

diisopropylfluorophophate irreversible inhibitor - forms covalent bond with active site of ser residue

Question 37

serine protease: p-nitrophenylacetate

Answer 37

is a product as the acyl enzyme forms . lag in acetate release following p-nitrophenolate release from the enzyme indicative of formation of an intermediate as well as the order or release of products

Question 38

Aspartic Proteases

Answer 38

involve two ASP residues in the active site - work together as general acid-base cat - one has a low pka and the other a high pka - h+ flow in rxn

Question 39

Aspartic Proteases : | protonated ASP

Answer 39

acts as general acid donates H+, facilitating formation of tetrahedral intermediate

Question 40

Aspartic Proteases: | deprotonated asp

Answer 40

acts as a general base accepting H+ from h2o forms OH- transition state

Question 41

Chorismate Mutase

Answer 41

cat rxn that can proceed w/o being cat. so we can compare because some cat rxn take years to occur - Claisen rearrangement - is the reaction of the enzyme

Question 42

Chorismate Mutase - transition state analogue

Answer 42

is stabilized by 12 electrostatic and H-bonds

Question 43

Chorismate Mutase - two way transition state can occur

Answer 43

it can be a boat or a chair | - most agree that it is a chair conformation

Question 44

Chorismate Mutase : mechanism

Answer 44

carboxyvinyl groups folds up and over the chorismate ring and the reaction proceeds via an internal rearrangement

Question 45

Chorismate Mutase : enzyme structure

Answer 45

its a homodimer - two equivalent active sites, each formed from portions of both monomers

Question 46

ribozyme group I intron

Answer 46

can splice it's self - a nuc. attacks and splices the intron and then it is activated and splices it's self with the help of cofactor guanosine.

Question 47

enzymes

Answer 47

can be protein or RNA

Question 48

engineered ribozymes

Answer 48

can cat. most known reactions although w/ lesser efficiency (kcat/Km) than is exhibited by protein enzymes