Investigators used the technique of site-directed mutagenesis to synthesize five

Question

Investigators used the technique of site-directed mutagenesis to synthesize five mutant creatine kinase proteins in which the Cys 278 was replaced with either a Gly, Ser, Ala, Asn or Asp residue. These mutants were termed C278G, C278S, C278A, C278N and C278D, respectively, to indicate the exact position of the amino acid change. The activities of the mutant enzymes were measured in the presence and absence of specific cofactors.

a. All of the mutants had decreased creatine kinase activity as compared to the wild-type enzyme. What information does this give you about the wild-type enzyme mechanism?

b. The activity of the mutant enzymes C278D and C278N were compared and it was found that the activity of the C278D mutant was 12-fold greater than the activity of the C278N mutant (although both mutants had lower enzyme activities as compared to the wild-type). Suggest an explanation for this observation.

c. The activities of the mutant enzymes (although decreased from the wild-type) were enhanced when either chloride or bromide ions were added to the assay mixture. (An exception was the C278D mutant). Why do you think that the ions were able to enhance enzyme activity?

d. The C278D mutant was an exception to the observation described in Question 2c above. This mutant did not show an enhancement of enzyme activity in the presence of chloride and bromide ions; in fact its minimal enzyme activity decreased somewhat in the presence of these ions. Explain why.

Explanation / Answer

a. This implies all the aminoacids are present in the active site of the enzyme and improtant for its activity. Mutating these residues directly impact its activity.

b. This increased activity of C278D being 12 fold greater than C278N mutant could be due to the presence of free COO- group present in the catalytic site of C278D mutant. The free COO- group could act as an electrophile to perform the catalytic mechanism compared to C278N mutant.

c. The chloride and bromide ions could act as a electron donor in the active site of the enzyme for the catalytic mechanism.

d. The C278D mutant having a Aspartate residue having a negatively charged side chain donates the electron for the catalysis, but when the addition of chloride and bromide ions the elctron donor groups are removed from the active site.

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