At 37 C, the serine protease subtilisin has k cat = 50 s1 and K M = 1.4×10 4 M.

ID: 55020 • Letter: A

Question

At 37 C, the serine protease subtilisin has kcat = 50 s1 and KM = 1.4×104 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 s1 and KM = 2×104 M.

1) 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.

Replace the methionine, by site-directed mutagenesis, with another residue. Because methionine is quite __, a ___ replacement would seem appropriate. A single base change in the Met codon could yield Phe, Leu, Ile, or __.

Word choices: hydrophilic, hydrophobic, Ser, His, Val

2) 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? Check all that apply.

The oxyanion is formed after S binds.

3) 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?

Express your answer to two significant figures and include the appropriate units.

4) The value you calculated in part 3 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: Coulomb's Law.

The dielectric constant is ___ in the enzyme active site than it is in water; thus, Coulomb's law predicts a __ interaction between the H-bond donor and acceptor.

Word choices: weaker, stronger, higher, lower

For a mutation of a residue that only interacts with the oxyanion intermediate, one would not expect KM to change significantly. For a mutation of a residue that only interacts with the oxyanion intermediate, one would expect KM to change significantly. kcat should be reduced due to the loss of enthalpic stabilization of the transition state. The oxyanion is formed after S goes away. kcat should be reduced due to the gain of enthalpic stabilization of the transition state.

The oxyanion is formed after S binds.

3) 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?

Express your answer to two significant figures and include the appropriate units.

Explanation / Answer

1).

Methionine is a non-polar, hydrophobic amino acid.

The site-directed mutagenesis is also called site-specific or oligonucleotide-directed mutagenesis. In this mutation a single base or multiple base changes, deletion or insertion can be done.

This technique is used to produce a protein with improved or special properties. The laundry detergent contains subtilisin. The wild-type of subtilisin has a methionine that can be oxidized by bleach, which reduces the activity of the protein. Thus, to enhance and maintain the protein activity methionine may be replaced by alanine or other hydrophobic residues. This modification helps the protein to be resistant to oxidation thereby keeping the protein active in the presence of bleach.

Replace the methionine, by site-directed mutagenesis, with another residue. Because methionine is quite hydrophobic, a hydrophobic replacement would seem appropriate. A single base change in the Met codon could yield Phe, Leu, Ile, or Val.

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At 37 C, the serine protease subtilisin has k cat = 50 s1 and K M = 1.4×10 4 M.

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