At 37 âC, the serine protease subtilisin haskcat = 50 sâ1 and KM = 1.4Ã10â4 M. Itis proposed that the N155 side chain contributes a hydrogen bond tothe oxyanion hole of subtilisin. J. A. Wells and colleaguesreported (1986, Phil. Trans. R. Soc. Lond. A 317:415-423)the following kinetic parameters for the N155T mutant ofsubtilisin: kcat = 0.02 sâ1 and KM =2Ã10â4 M.
1) Subtilisin does have a problem in that it becomes inactivatedby oxidation of a methionine close to the active site. Suggest away to make a better subtilisin.
Replace the methionine, by site-directed mutagenesis, withanother residue. Because methionine is quite __, a ___ replacementwould seem appropriate. A single base change in the Met codon couldyield Phe, Leu, Ile, or __.
Word choices: hydrophilic, hydrophobic, Ser, His, Val
2) Is the effect of the N155T mutation what you would expect fora residue that makes up part of the oxyanion hole? How do thereported values of kcat and KM support youranswer? Check all that apply.
For a mutation of a residue that only interacts with theoxyanion intermediate, one would not expect KM to changesignificantly. For a mutation of a residue that only interacts with theoxyanion intermediate, one would expect KM to changesignificantly. kcat should be reduced due to the loss of enthalpicstabilization of the transition state. The oxyanion is formed after S goes away. kcat should be reduced due to the gain of enthalpicstabilization of the transition state. The oxyanion is formed after S binds.
3) Assuming that the T155 side chain cannot H-bond to theoxyanion intermediate, by how much (in kJ/mol) does N155 appear tostabilize the transition state at 37 âC?
Express your answer to two significant figures and include theappropriate units.
4) The value you calculated in part 3 represents the strength ofthe H-bond between N155 and the oxyanion in the transition state.This value is higher than typical H-bonds in water. How might thisobservation be rationalized? Hint: Coulomb's Law.
The dielectric constant is ___ in the enzyme active site than itis in water; thus, Coulomb's law predicts a __ interaction betweenthe H-bond donor and acceptor.
Word choices: weaker, stronger, higher, lower
At 37 âC, the serine protease subtilisin haskcat = 50 sâ1 and KM = 1.4Ã10â4 M. Itis proposed that the N155 side chain contributes a hydrogen bond tothe oxyanion hole of subtilisin. J. A. Wells and colleaguesreported (1986, Phil. Trans. R. Soc. Lond. A 317:415-423)the following kinetic parameters for the N155T mutant ofsubtilisin: kcat = 0.02 sâ1 and KM =2Ã10â4 M.
1) Subtilisin does have a problem in that it becomes inactivatedby oxidation of a methionine close to the active site. Suggest away to make a better subtilisin.
Replace the methionine, by site-directed mutagenesis, withanother residue. Because methionine is quite __, a ___ replacementwould seem appropriate. A single base change in the Met codon couldyield Phe, Leu, Ile, or __.
Word choices: hydrophilic, hydrophobic, Ser, His, Val
2) Is the effect of the N155T mutation what you would expect fora residue that makes up part of the oxyanion hole? How do thereported values of kcat and KM support youranswer? Check all that apply.
| For a mutation of a residue that only interacts with theoxyanion intermediate, one would not expect KM to changesignificantly. | |
| For a mutation of a residue that only interacts with theoxyanion intermediate, one would expect KM to changesignificantly. | |
| kcat should be reduced due to the loss of enthalpicstabilization of the transition state. | |
| The oxyanion is formed after S goes away. | |
| kcat should be reduced due to the gain of enthalpicstabilization of the transition state. | |
| The oxyanion is formed after S binds. 3) Assuming that the T155 side chain cannot H-bond to theoxyanion intermediate, by how much (in kJ/mol) does N155 appear tostabilize the transition state at 37 âC? Express your answer to two significant figures and include theappropriate units. 4) The value you calculated in part 3 represents the strength ofthe H-bond between N155 and the oxyanion in the transition state.This value is higher than typical H-bonds in water. How might thisobservation be rationalized? Hint: Coulomb's Law. The dielectric constant is ___ in the enzyme active site than itis in water; thus, Coulomb's law predicts a __ interaction betweenthe H-bond donor and acceptor. |
