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