Problem:

Sulfhydryl groups have the ability to react with the alkylating reagent N-ethylmaleimide (NEM) in chemical modification experiments. When NEM is added to a purified solution of creatine kinase, Cys 278 is alkylated, but no other Cys residues in the protein are modified.

Question: What can you infer about the Cys 278 residue based on this observation?

Based on the result described in Question 1, the 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.

Required:

Question 1: 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?

Question 2: 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.

Question 3: 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?

Question 4: 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.

Explain all the answers in detail.