At 37 °C, the serine protease subtilisin has kcat = 50 s-1 and KM = 1.4 × 10-4 M. It is proposed that the N155 side chain contributes ahydrogen bond to the oxyanion hole of subtilisin. J. A. Wells and colleagues reported (1986, Phil. Trans. R. Soc. Lond. A 317:415–423) the following kinetic parameters for the N155T mutant of subtilisin: kcat = 0.02 s-1 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 abetter subtilisin.(c) Is the effect of the N155T mutation what you would expect for a residuethat makes up part of the oxyanion hole? How do the reported values ofkcat 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 thetransition state at 37 °C?(e) The value you calculated in part (d) represents the strength of theH-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?
Enzyme kinetics
In biochemistry, enzymes are proteins that act as biological catalysts. Catalysis is the addition of a catalyst to a chemical reaction to speed up the pace of the reaction. Catalysis can be categorized as either homogeneous or heterogeneous, depending on whether the catalysts are distributed in the same phase as that of the reactants. Enzymes are an essential part of the cell because, without them, many organic processes would slow down and thus will affect the processes that are important for cell survival and sustenance.
Regulation of Enzymes
A substance that acts as a catalyst to regulate the reaction rate in the living organism's metabolic pathways without itself getting altered is an enzyme. Most of the biological reactions and metabolic pathways in the living systems are carried out by enzymes. They are specific for their works and work in particular conditions. It maintains the best possible rate of reaction in the most stable state. The enzymes have distinct properties as they can proceed with the reaction in any direction, their particular binding sites, pH specificity, temperature specificity required in very few amounts.
At 37 °C, the serine protease subtilisin has kcat = 50 s-1 and KM = 1.4 × 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 colleagues reported (1986, Phil. Trans. R. Soc. Lond. A 317:415–423) the following kinetic parameters for the N155T mutant of subtilisin: kcat = 0.02 s-1 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
kcat 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?
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