(Y409). (D149) OH OH H,N, R374 R' H,Ñ Но OR 1st Step (E117) ROH (Y409). (D149) OH OH R374 R" H,Ñ H,0. Но (E117) 2nd Step (Y409). (D149) ö: он OH H,N C-N, coo H,N R' R374 но (E117) 3rd Step (Y409). (D149) OH OH HÖ H,N, co H,N R" R374 но OH (E117) но
Shown below is a proposed mechanism for the cleavage of sialic acid by the viral enzyme neuraminidase. The kcat for the wild-type enzyme at pH =
6.15, 37 °C 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 Glu 117 to Ala to have a greater effect on KM or kcat?
(c) For the R374N mutant at pH = 6.15, 37 °C, kcat is 0.020 s-1, and KM
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?).
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