Chapter 8, Problem 24P

In kinetics experiments, the hydrolysis of the substrate sialic acid by neuraminidase appears to obey Michaelis—Menten kinetics. Neuraminidase activity is critical for viral infectivity; thus, this enzyme is the target of much work by pharmaceutical companies to develop a drug to treat influenza virus
infection. The drug "Tamiflu" is a competitive inhibitor of neuraminidase. Initial rate data collected at
pH = 6.15, 37 ^oC with 0.021 µM neuraminidase and 25.0 µM sialic acid gives a Lineweaver—Burk plot with a slope of 51.2 s.
a. Recall from Problem that the k_cat for neuraminidase at pH = 6.15, 37 ^oC is 26.8 s-1. Calculate K_M for the hydrolysis of sialic acid.
b. When the reactions in part (a) are repeated in the presence of 0.040 µM of Tamiflu, the slope of the Lineweaver—Burk plot is 198.8 s. Calculate the value of K_I for Tamiflu.

23. Shown below is a proposed mechanism for the cleavage of sialic acid by the viral enzyme
neuraminidase. The k_cat for the wild-type enzyme at pH = 6.15, 37 ^oC is 26.8 s-1.
a. Describe the roles of the following amino acids in the catalytic mechanism: Glu117, Tyr409, and Asp149. List all of the following that apply: general acid/base catalysis (GABC), covalent catalysis, electrostatic stabilization of transition state.
b. Based on the information shown in the scheme, would you expect mutation of Glu117 to Ala to have a greater effect on K_M or k_cat?
c. For the R374N mutant at pH = 6.15, 37 ^oC, k_cat is 0.020 s-1, and K_M is relatively unaffected. Based on this result, it seems that R374 is more critical for catalysis than for substrate binding.
Explain how R374 stabilizes the reaction transition state more than the substrate (i.e., what feature of this reaction would explain tighter binding to the transition state vs. substrate?).