At 37 °C, the serine protease subtilisin has kat = 50 s1 and KM= 1.4 x 10-4 M. It is proposed that the N155 side chain contributes a hydrogen bond to the oxyanion hole of subtilisin. J. A. Wells and col- leagues reported (1986, Phil. Trans. R. Soc. Lond. A 317:415–423) the following kinetic parameters for the N155T mutant of subtilisin: kat = 0.02 s-' and KM = 2 × 10-4 M. (a) Subtilisin is used in some laundry detergents to help remove protein-type stains. What unusual kind of stability does this suggest for subtilisin? (b) Subtilisin does have a problem, in that it becomes inactivated by oxidation of a methionine close to the active site. Suggest a way to make a better subtilisin. (c) Is the effect of the N155T mutation what you would expect for a residue that makes up part of the oxyanion hole? How do the reported values of kat and KM support your answer? (d) Assuming that the T155 side chain cannot H-bond to the oxyanion intermediate, by how much (in kJ/mol) does N155 appear to stabilize the transition state at 37 °C? (e) The value you calculated in part (d) represents the strength of the H-bond between N155 and the oxyanion in the transition state. This value is higher than typical H-bonds in water. How might this observation be rationalized? Hint: consider Equation 2.2 (Coulomb's Law).
At 37 °C, the serine protease subtilisin has kat = 50 s1 and KM= 1.4 x 10-4 M. It is proposed that the N155 side chain contributes a hydrogen bond to the oxyanion hole of subtilisin. J. A. Wells and col- leagues reported (1986, Phil. Trans. R. Soc. Lond. A 317:415–423) the following kinetic parameters for the N155T mutant of subtilisin: kat = 0.02 s-' and KM = 2 × 10-4 M. (a) Subtilisin is used in some laundry detergents to help remove protein-type stains. What unusual kind of stability does this suggest for subtilisin? (b) Subtilisin does have a problem, in that it becomes inactivated by oxidation of a methionine close to the active site. Suggest a way to make a better subtilisin. (c) Is the effect of the N155T mutation what you would expect for a residue that makes up part of the oxyanion hole? How do the reported values of kat and KM support your answer? (d) Assuming that the T155 side chain cannot H-bond to the oxyanion intermediate, by how much (in kJ/mol) does N155 appear to stabilize the transition state at 37 °C? (e) The value you calculated in part (d) represents the strength of the H-bond between N155 and the oxyanion in the transition state. This value is higher than typical H-bonds in water. How might this observation be rationalized? Hint: consider Equation 2.2 (Coulomb's Law).
Chemistry
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ISBN:9781305957404
Author:Steven S. Zumdahl, Susan A. Zumdahl, Donald J. DeCoste
Publisher:Steven S. Zumdahl, Susan A. Zumdahl, Donald J. DeCoste
Chapter1: Chemical Foundations
Section: Chapter Questions
Problem 1RQ: Define and explain the differences between the following terms. a. law and theory b. theory and...
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