The investigators next carried out kinetic studies in which they measured the ab

Question

The investigators next carried out kinetic studies in which they measured the ability of a second substrate to bind once the first substrate had bound. The data are shown in Table 15.1. Kd refers to the constant for the binding of substrate to free enzyme and KM refers to the constant for binding of that same substrate to the enzyme when the other substrate is already bound to the enzyme. The results for the wild type and two of the mutants are shown in Table 15.1.

b.Similarly, compare the Kd and KM values for the two mutant enzymes. Again, what does a comparison of the Kd and KM values tell you about the ability of each substrate to bind to the enzyme alone? to the enzyme when the other substrate is present?

c. Consider your answers to 4a and 4b and considerl the Vmax valdes mutant enzymes. Assess the role of Cys 278 in the binding of creatine and ATP substrates to the creatine kinase enzyme. Table 15.1: Binding constants for phosphocreatine synthesis from creatine and ATP (modified from Furter, et al., 1993). Creatine ATP max Wild-type19.6 C278G C278S 8.9 273 209 0.70 0.27 0.31 0.32 1.13 0.70 60.7 6.0 2.0 64 92 eference urter, R., Furter-Graves, E. M., and Wallimann, T. (1993) Biochemistry 32, pp. 7022-7029.

Explanation / Answer

a) Low K value signifies high substrate affinity and vice-versa. For both creatine and ATP, Kd value is higher than Km. When substrate, creatine binds to the wild type free enzyme, Kd value comes out to be 19.6mM. This value is high which means that wild type enzyme will have low affinity for creatine. When the same substrate, creatine binds to a second site of wild type enzyme, Km value comes out to be 8.9mM. This value is lower than Kd. So, after binding of second substrate, now the wild type enzyme will have greater affinity for creatine. Initially, the wild type free enzyme had low affinity for binding to the substrate, creatine; and after binding of creatine, the affinity of binding of free enzyme increases.

               Same is the case for ATP.

b) The condition reverses in case of mutant enzymes.

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