lecture 46 - enzyme catalysis mechanisms

Question 1

describe the catalytic relationship between enzymes and the transition state in terms of ΔG

Answer 1

enzymes decrease the ΔG of the transiton state by some amount ΔGb (binding)

Question 2

what are the three modes of catalysis?

Answer 2

(1) acid/base catalysis
(2) metal ion catalysis
(3) covalent catalysis

Question 3

describe acid/base catalysis

Answer 3

• donation/abstraction of an H+
• causing an increase in electrophilicity (donating a proton) or in nucleophilicity (if abstracting a proton)
• or stabilization of a charge (often T state produces charges

Question 4

describe metal ion catalysis

Answer 4

• positive metal ions stabilize the negative charges formed in the transition state
• may also take part in redox reactions

Question 5

describe covalent catalysis

Answer 5

• fomration of transient covalent bonds between the enzyme and susbstrate
• this creates an alternate reaction pathway

Question 6

explain (generally) the hydrolysis of a peptide bond

Answer 6

-have protein in 6M HCl
-water and hydronium ions interact with the peptide bond to form a transition state molecule where bonds are breaking and reforming and partial charges
-next the molecule form a tetrahedral intermediate, then another intermediate, until forming the final product:
X1 – C – OH + NH2 – X2
||
O

Question 7

what accelerates the hydrolysis of a peptide bond?

Answer 7

H+ transfer from the hydronium ion to stabilize the negative charge that would form on the oxygen of the carbonyl in the transition state/tetrahedral intermediate accelerates the reaction

Question 8

how could an enzyme accelerate the hydrolysis of a peptide bond under mild conditions? (6)

Answer 8

(1) protonate side chain in place of h3o+ (donate h+)
(2) protonate side chain that forms an ionic bond (interact with neg charge on carbonyl oxygen)
(3) use positively charged metal ion to stabilize the neg charge on the oxygen
(4) use a base to activate h2o as a neucelophile
(5) covalent catalysis (diff rxn pathway)
(6) proximity and orientation effects (substrate is bound such that the enzymes active site groups are optimally positioned)

Question 9

give examples of serine proteases

Answer 9

chymotrypsin, trypsin (homologues)

Question 10

describe the active site cleft of serine proteases

Answer 10

• binds a polypeptide substrate
• includes a “recognition pocket” - orients the substrate so that the carbonyl carbon is close to a Ser-OH
• i.e. interaction does not happen at the recognition pocket, happens close by

Question 11

what does chymotrypsin bind?

Answer 11

aromatics (F, W, Y)

Question 12

what does trypsin bind?

Answer 12

K and R

Question 13

what is the catalytic triad for serine-proteases?

Answer 13

serine h-bonded to a histidine which is h-bonded to an aspartate (which has a neg charge)

Question 14

how does the catalytic triad arise?

Answer 14

the 3D structure of the enzyme positions the residues side chains close together so that they can h-bond

Question 15

what is an important feature of the catalytic triad?

Answer 15

• the asp is burried (inaccessible to solvent)
• means it can’t be protonated from water
• also means negative charge can be distributed
• makes the asp a stronger base - means it more liekly to take up a proton (pKa increases)

Question 16

describe the His—Asp h-bond
why is this bond this way?
what does this bond allow for?

Answer 16

• very short and very strong
• this is because of the neg charge on asp
• allows his to be a stronger base (pKr=12)
• this allows his to pull an H+ off of the Ser-OH

Question 17

describe the Ser-O(-) product and why it is important for the catalytic function of serine enzymes

Answer 17

• much stronger nucelophile than Ser-OH

- can better attack the carbonyl group of the substrate

Question 18

describe the actual mechanism by which serine proteins function

Answer 18

(1) catalytic triad binds the peptide
-asp activates his
-his then acts as a base
-ser is a nucelophile
(2) formation of a h-bond btwn the serine O and the carbonyl carbon and the conversion of the carbonyl carbon from planar to a tetrahedral intermediate
-his is now protonated and acts as an acid (donates H+ to the leaving group)
(3) covalent catalysis
-now have acetyl-enzyme intermediate
-the leaving group exits the active site
-h-bond btwn his and ser is broken
(4) His acts as a base to deprotonate h2o, activating it so it can act as a nucleophile, causing the formation of the 2nd tetrahedral intermediate
(5) His acts as an acid to form the pro, reforming the h-bond
(6) catalytic triad is restored and the product leaves the active site OH
/
product: NH – C = O
|
R2

Question 19

how do the actions of serine proteases decrease the ΔG of the transition state? (3)

Answer 19

(1) proximity and orientation
(2) acid base catalysis
(3) electrostatic stabilization of the transition state and of tetrahedral intermediates

Question 20

describe how proximity and orientation contribute to a decreased ΔG of the transition state

Answer 20

• substrate binds the enzyme such that Ser-O(-) is in position to attack the carbonyl carbon
• His is positioned to act as a base and an acid
• h2o is positioned for attack
• binding energy contributes to this prositioning and orientation of various groups