lecture 47 - transition state stabilization and substrate recognition

Question 1

what is the primary way in which enzymes make reactions go faster?

Answer 1

stabilization of the transition state

Question 2

give a general reaction for the conversion of a substrate into its products via an enzyme
then show the gibbs free energy graph

Answer 2

E + S = ES = EP* + Q = EP + Q = E + P + Q

• small bump from E+S–>ES
• bigger bump with two peaks (tetrahedral intermediate in the center of the peaks) from ES –> EP*
• another bigger bump with two peaks tetrahedral intermediate in the center of the peaks) from EP* –> EP
• small bump from EP –> E + P + Q
• recall: bump = transition state

Question 3

are tetrahedral intermediates higher or lower in energy? why?

Answer 3

higher in energy due to the formation of charge

Question 4

how do serine proteases stabilize the transition state and tetrahedral intermediates without stabilising ES too much? (2)

Answer 4

(1) the partial negative charge on the carbonyl O is stabilized by 2 h-bonds to 2 backbone NH groups (gly and ser ** same ser from the catalytic triad)
(2) the conformaiton of the carbonyl group to C-O(-) causes a change from planar to tetrahedral and from a short c=o bond to a longer c-o bond

Question 5

describe in more detail the effects of the conformation of the C=O to C-O(-)

Answer 5

• the O from the carbonyl shift position as the nucleophile attacks to form the tetrahedral intermediate
• also a the same time, the O gains a negative charge (becomes an oxyanion)
• this leads to the formation of the oxyanion hole
• i.e. the oxyanion forms h-bonds only to the transition state and the tetrahedral intermediate
• this results in preferential stabilization of the transiton state/tetrahedral intermediates
• therefore, decreasing the ΔG of the transition state

Question 6

what does the enzyme use to position the substrate for reactions?

Answer 6

binding energy

Question 7

what does the enzyme provide to stabilize the transition state? (3)

Answer 7

• enzymes provide interactions that only occur in the transition state or in high energy intermediates
• enzymes provide electrostatic stabilization of the charges found in the transition state/tetrahedral intermediate
• some enzymes add strain to the substrate to distort its conformation towards the transition state structure, decreasing strain as the transition state forms

Question 8

describe the specificity of chymotrypsin

Answer 8

• cuts after aromatics (F,W,Y)
• has a large, deep, and mostly hydrophobic pocket as the regocnition domain for aromatics
• when the peptide is bound to the enzyme it is in the beta conformation (extended backbone conformation)
• then the enzyme cleaves the peptide after the aromatic residue
• “R2” is not recognized, where R2 = the following aa side chain

Question 9

describe the specificity of trypsin

Answer 9

• cuts after R, K
• has a large deep pocket with a negative charge at the bottom
• when the peptide is bound to the enzyme it is in the beta conformation (extended backbone conformation)
• then the enzyme cleaves the peptide after the pos residue
• “R2” is not recognized, where R2 = the following aa side chain

Question 10

describe the specificity of elastase

Answer 10

• cuts after small reisdues (S, V, T)

- has deep but narrow pocket compared to trypsin

Question 11

why are the recognition pockets different for serine proteases and elastase?

Answer 11

• because of an aa substitution
• pocket of serine proteases is made by 2 gly residues
• pocket of elastase subsititues gly for val and thr

Question 12

why don’t trypsin and chymotrypsin cut after small aa residues?

Answer 12

because there are fewer optimal reactions (bc of smaller side chain):

• this decreases binding affinity (decreases recognition of the substrate)
• this also leads to a less optimal position in the transition state

Question 13

all interactions between the enzyme and the substrate/transition state are important for what?
what does this mean for the transition state binding?

Answer 13

• positioning

- therefore the more precise the fit of enzyme and the transition state results in a decrease in activation energy

Question 14

can proteases recognize longer sequences?

Answer 14

yes

Question 15

what type of protease are TEV proteases?

Answer 15

cys proteases

Question 16

what composes the catalytic traid of TEV proteases?

Answer 16

cys-his-arg

Question 17

what do TEV proteases recognize/cleave?

Answer 17

• cleaves polyproteins at multiple sites
• recognizes a sequence pattern: E, X, L, Y, Φ, Q // Ψ
• X = any aa
• Φ is a medium/large hydrophobic aa
• Ψ is a small hydrophobic or planar aa
• // = the cleavage site

Question 18

descirbe the active site interactions of TEV proteases

Answer 18

• binding site has specific interactions for 7 residues of a substrate (E, X, L, Y, Φ, Q // Ψ)
• interactions at the binding site are most specific for E, L, Y, Q

Question 19

what is a unique feature of TEV proteases?

Answer 19

• can recognize chiral substrate
• if the protese has a specificity pocket to bind with a group ‘R’, and if the R group is positioned into the page:
• the aa cannot position the backbone properly and fit the R-group into the pocket at the same time - so it won’t bind