Test 2 Ch. 9 Catalytic Strategies

Front 1

Describe a ping-pong double displacement reaction

Back 1

One or more products are released before all substrates

1st substrate binding --> 1st release, 2nd substrate binding --> second released

Front 2

Describe an ordered sequential reaction

Back 2

All reactants bind in before the reaction occurs

Front 3

What reaction type is defined by the existence of a substituted enzyme intermediate?

Back 3

Ping-pong reaction

Front 4

How does a complementarity pocket contribute to catalysis?

Back 4

Complementarity pocket stabalizes the substrate

Front 5

What are the 6 catalytic mechanisms of enzymes?

Back 5

1) Proximity and orientation

2) preferential binding of the transition state complex

3) Catalysis by destabalization of substrate

4) Acid-base catalysis

5) Covalent catalysis (E+ and Nu: catalysis)

6) Metal Ion catalysis

Front 6

What side chains are commonly used in acid-base catalysis?

Back 6

AA's with pK's in the physiological range

Asp, Glu, His, Cys, Tyr, and Lys

Front 7

What AA's are common nucleophiles?

Back 7

Histidine's imidazole group

Unprotonated Lys

Thiol of Cys

Carboxyl group of Asp

Hydroxyl of Ser

Front 8

What are common electrophiles from AA groups?

Back 8

Carbonyl

Protonated Imine C=NH+

Phosphorous

Front 9

What amino acids make up the catalytic triad?

Back 9

Asp, His, and Ser

Front 10

What is the role of the Asp in the catalytic triad?

Back 10

Hydrogen bond donor to N-H of Imidazole to stabalize charged His, but doesn't actually take the His proton

Front 11

What is the role of His in the catalytic triad?

Back 11

H+ acceptor to serine and water increasing it's oxygens Nu: towards the carbonyl

H+ donor to tetrahedral intermediate to yield product

Hydrogen bond donor to intermediate to yield free amine component

Acid-Base catalysis

Front 12

How does destabalization facilitate catalysis?

Back 12

Energy of substrate is brought closer to transition state energy and the stress is then relieved at the transition state

Front 13

Destabalization by desolvation is a method of catalytic substrate destabalization, how does it work?

Back 13

Remove a very polar functional group from water raising its energy and making it more reactive

Front 14

Chymotrypsin utilizes 3 catalytic strategies, what are they?

Back 14

Nu: catalysis, stabilization of the transition state, general acid/base

Front 15

Why do we observe 2 phases in chymotypsins action?

Back 15

The first step is faster due to covalent binding of ES intermediate while the second step is slower since it's hydrolysis

Front 16

What is stage 1 of chymotrypsin cleavage?

Back 16

Substrate binding --> Nu: attack of serine on the peptide carbonyl --> collapse of the tetrahedral intermediate by protonation of amino group--> Release of amine component

Front 17

What is stage 2 of chymotrypsin cleavage?

Back 17

Water binding --> Nu: attack of water on the acyl-enzyme intermediate by His activation general base catalysis--> Collapse of 2nd tetrahedral intermediate --> Release of the carboxylic acid component --> Reprotonation of Set195 by His

Front 18

What methods of catalysis does Carbonic anhydrase use?

Back 18

Electrophilic, metal ion

Front 19

What is the difference between random and ordered sequential mechanism's initial binding?

Back 19

Random: Either substrate or coenzyme can bind first to enzyme before catalysis
Ordered: Coenzyme must bind before substrate can and then subsequent catalysis

Front 20

What is the difference between random and ordered sequential mechanism's product release?

Back 20

Random: Either product or coenzyme can be released first
Ordered: Product must be released before coenzyme

Front 21

What is the defining characteristic of a double–displacement (ping–pong) reaction?

Back 21

The existence of a temperature modified substituted enzyme intermediate

Front 22

What is the general mechanism of a ping–pong reaction?

Back 22

First substrate binding ––> First product release, Second Substrate binding ––> Second product Release

Front 23

The difference in free energy between substrate and transition state is indirectly proportional to _______

Back 23

The concentration of the transition state!

The smaller the diff. in free energy, the greater concentration of transition state

Front 24

What feature of an enzyme allows for the destabilization of substrate?

Back 24

An enzyme pocket complementary to the rxn transition state

Front 25

How does an enzyme compensate for the energy required to destabilize a substrate?

Back 25

The binding energy compensates

Front 26

What happens to the activation energy if the enzyme is complementary to the substrate?

Back 26

The ES complex is more stable and has less free energy in the ground state than the substrate does alone leading to an increase in activation energy

Front 27

What are the 6 catalytic mechanisms of enzymes discussed in lecture?

Back 27

1) Proximity and Orientation Effects
2) Preferential binding of the transition state complex
3) Catalysis by destabilizing the substrate
4) Acid–base catalysis
5) Covalent (Electrophilic of Nu:) catalysis
6) Metal Ion catalysis

Front 28

What side chains are commonly used in acid–base catalysis and what feature of their pK allows this?

Back 28

1) Asp
2) Glu
3) His
4) Cys
5) Tyr
6) Lys All have pK's in the physiological range

Front 29

What AA's commonly act as Nu:?

Back 29

1) Imidazole group of His
2) Unprotonated amino group of Lys
3) Thiol of Cys
4) Carboxyl of Asp
5) Hydroxyl of Ser

Front 30

What are common cofactors?

Back 30

1) Fe 2+ and 3+
2) Cu 2+
3) Mn 2+
4) Co 2+
5) Mg 2+
6) Zn 2+

Front 31

What are common electrophiles?

Back 31

Carbon atom of a carbonyl
Protonated Imine carbon
Phosphorous of a phosphate group

Front 32

What type of catalytic mechanism does Complex III use and why is it characterized like that?

Back 32

Proximity and Orientation effects

Its preorients cytochrome C and subunit cytochrome C1 of complex III for heme–to–heme e– transfer

Front 33

What type of catalytic mechanism does the catalytic triad of serine protease use and why is it characterized like that?

Back 33

Catalysis by preferential binding of the transition state because Ser195's Nu: attach on the scissile peptide carbonyl leads to stronger binding by filling the oxyanion hole and hydrogen bonding between backbone NH and CO of Gly193 which it was unable to do prior to its transition state

Front 34

What are the 3 types of substrate destabilization and what do they all have in common

Back 34

1) Entropy reduction
2) Desolvation
3) Stress
Destabilization raises the free energy of the substrate bringing it closer to the transition state

Front 35

Describe how an enzyme can contribute to entropy reduction as a method of substrate destabilization

Back 35

When Substrate binds enzyme, there are reduced degrees of freedom available for translational motion leaded to a more ordered lower entropy situation
This increases its free energy ––> more reactive

Front 36

Describe how an enzyme can contribute to desolvation as a method of substrate destabilization

Back 36

The substrate can only bind without it's polar solvation shell to the enzyme which raises it's energy and makes it more reactive

Front 37

What types of "stress" can destabilize a substrate and aid in catalysis?

Back 37

Electrostatic or steric
Similar charged in proximity raises the free enrgy making it more reactive

Front 38

What's the difference between specific and general acid–base catalysis?

Back 38

Specific involves the diffusion of H+ or OH– into the catalytic center
General involves acids other than H+ and bases other than OH–

Front 39

in what ways can a metal ion aid in electrophilic catalysis?

Back 39

1) Can increase susceptibility to Nu: attach by making it more electron withdrawn
2) By making the leaving group a weaker base aka stabilizing LG's charges
3) By increasing Nu: of water by facilitating leaving of proton and just leaving a metal–bound OH

Front 40

How can metal ion cofactors aid in catalysis?

Back 40

1) To shield of stabilize negative charges
2) To mediate Redox through changes in the oxidation state
3) To bind and orient substrates

Front 41

How does a zymogen regulate chymotrypsins activity?

Back 41

Chymotrypsin is synthesized as 1 polypeptide chain called chymotrypsinogen which is cleaved and activated by zymogen

Front 42

How is the zymogen that regulates chymotrypsin regulated?

Back 42

Trypsinogen is cleaved and activated to trypsin which leads to a protease cascade eventually cleaving and activating chymotrypsin

Front 43

How does Dicoumoral inhibit proper prothrombin synthesis?

Back 43

Dicoumoral is a vitamin K antagonist like warfarin.

Vitamin K is essential to proper synthesis, so it's inhibition leads to abnormal prothrombin that cannot bind calcium ion

Front 44

What is the role of proteolytic cleavage in the formation of a blood clot?

Back 44

Prothrombin must be proteolytiaclly cleaved to thrombin which cleaves fibrinogen to fibrin which polymerizes with fibrinopeptides which becomes a clot!

Front 45

How does fibrin polymerize to form a clot?

Back 45

Glutamate residues on one chain react with lysine residues on another and their formation is catalyzed by transglutaminase

Front 46

What is the binding scheme and function of Zinc ion in carbonic anhydrase catalysis?

Back 46

Zn2+ is always bound to 4 ligands, 3 His and 1 H2O

The Zn2+ allows water to lose a proton at neutral pH, making it a stronger Nu: to attack CO2

Front 47

Describe the Carbonic Anhydrase mechanism

Back 47

1) Zinc ion facilitates formation of OH from H2O, H+ is taken by proton shuttle

2) CO2 binds active site and is Nu: attacked by Zn2+ bound OH converting CO2 to HCO3- ion

3) Displacement of HCO3- by H2O and regeneration of active site

Front 48

What is the function of His 64 in carbonic anhydrase catalysis?

Back 48

His 64 shuttles protons form water to the surface of the protein to be picked up by the buffer

Front 49

What happens if we're missing a bufffer in carbonic anhydrase?

Back 49

Without a buffer the Kcat becomes limited to proton diffusion and slows down dramatically

Front 50

What are the roles of His96, His119, and His94 in Carbonic Anhydrase catalysis

Back 50

Binding site for Zinc Ion

Front 51

What methods of catalysis do restriction enzymes use?

Back 51

1) Electrophilic catalysis

2) Metal ion catalysis

3) Substrate distortion

Front 52

What are Type II restriction enzymes used for?

Back 52

Cloning and Sequencing DNA

They generate a 3' OH and 5' phosphate

Front 53

What is the cofactor for Type II restriction enzymes and what is it's purpose

Back 53

Mg2+

1) Distorts the substrate (Kink the TA DNA pairs)

2) Increases Nu: of water

Front 54

How does magnesium contribute to Type II restriction enzyme specificity?

Back 54

Only cognate sequence bind to and are distorted by Mg2+

Distortion of cognate complexes is offset by the increased binding energy associated with the Mg2+ - DNA complex.

Noncognate sequences cannot achieve full binding energy and so distortion is not offset

Front 55

How does water hydrolyze a cognate DNA sequence?

Back 55

In-line single displacement

Activated water attacks phosphorous as a Nu: and leads to inversion of stereochem via a pentacoordinate intermediate

Front 56

Where do a Myosin get its energy for conformational chagnes?

Back 56

Hydrolysis of Mg2+ - ATP complex

Front 57

What catalytic mechanism does ATP hydrolysis utilize?

Back 57

General Acid/Base

Front 58

What is the function of Mg2+ in ATP hydrolysis?

Back 58

ATP:Mg2+ is the substrate

Mg2+ is not a component of the active site

Mg2+ increases susceptibility of bond cleavage by withdrawing electron density

Front 59

What about ATP hydrolysis is unique in the Myosin active site?

Back 59

ATP hydrolysis is freely reversible, thus each ATP is cleaved and reformed several times before the products are released

Front 60

What is the mechanism that enables Myosin to change conformation?

Back 60

1) Hydroxide is formed from water through deprotonation by Ser 236, and the resulting OH- attacks the gamma-phosphoryl group --> This causes the ether bonds to break between the beta and gamma phosphoryl

2) Ser 236 is then deprotonated by the Gamma-phophoryl oxygen

Front 61

What is the P loop?

Back 61

Characteristic loop between first beta strand and first helix of the NTP-binding core that interacts with phosphoryl groups on the bound nucleotide