Biochemistry of the cell Spring 2013

An assignment modified from Site-directed mutagenesis of creatine kinase, from

Cases in Biochemistry, by K. Cornely

A technique called site-directed mutagenesis can be used to change single amino

acids in a protein. A particular example of this is changing selected amino acid
residues in the active site of the protein creatine kinase.

Creatine kinase (CK) phosphorylates creatine to form phosphocreatine. The reaction

is readily reversible, and can be assayed by measuring production of H+ (forward
reaction), or decrease in H+ concentration (reverse reaction).

This assignment is based on two techniques for the study of enzyme, the
aforementioned site-directed mutagenesis, and the covalent modification of amino
acid residues in the protein. For the latter, reagents are used that can irreversibly
modify a specific amino acid. The experimenter then measures the activity of the
enzyme, which is followed by sequencing of the protein to determine which residue
was modified. If a chemical modification results in inactivation of the protein, it can
be inferred that that particular amino acid is essential for the enzyme’s activity in
some way.

Here’s the reaction catalyzed by CK:

Here are the results of three chemical modification experiments performed with CK:

Questions
1. What can you infer about the Cys278 residue based on the following observation:
When N-ethylmaleimide is added to a solution of purified creatine kinase Cys278 is
alkylated (as shown above), but no other Cys residues in the protein are modified.
2. Based on results described in question 1, investigators used the technique of site-
directed mutagenesis to synthesize five mutant CK proteins in which the Cys278
residue was replaced with either a Gly, Ser, Ala , Asn or Asp residue. The mutants
were called C278G, C278S, C278A, C278N and C278D, respectively. The activities of
these enzymes were assayed.
All of the mutants had decreased creatine kinase activity as compared to the
wild-type enzyme. What information does this result provide about the reaction
mechanism in the wild-type enzyme?
The activity of the mutant enzyme C278D was 12-fold greater than the
activity of the C278N mutant. Suggest an explanation for this result.

3. Some investigators have modified Cys278 with iodoacetamide and found that the
enzyme activity was abolished. They concluded that Cys278 was essential for
enzymatic activity. Other investigators modified Cys278 with iodacetate and found
that enzyme activity was reduced, but not abolished, leading them to conclude that
Cys278 was not essential. Can you suggest an explanation that is consistent with
these observations and with your answer to question 2?

4. The investigators carried out kinetic studies in which they measured the ability of
a second substrate to bind once the first substrate had bound to the enzyme.
Compare the Kd and KM values for creatine and ATP for the wild-type
enzyme. What does this comparison tell you about the ability of each substrate to
bind to the enzyme alone, and when the other is present?
Make the same comparison for the two mutant enzymes.
Considering the two previous questions, and the values for Vmax, assess the
role of Cys278 in the binding of creatine and ATP to CK.

Enzyme Creatine Creatine ATP ATP Vmax

Kd (Mm) KM (Mm) Kd (Mm) KM (Mm) (µmol/min)
Wild-type 19.6 8.9 0.70 0.32 60.7
C278G 64 273 0.27 1.13 6.0
C278S 92 209 0.31 0.70 2.0