Catalysis by Two Sialidases with the Same Protein Fold but Different Stereochemical Courses: A Mechanistic Comparison of the Enzymes from Influenza A Virus and Salmonella typhimurium

Xumin Guo, W. Graeme Laver, Eric Vimr, Michael L. Sinnott

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Abstract

The protein folds and presumed active site residues of influenza A (Varghese, J. N., McKimm-Breschkin, J. L.; Caldwell, J. B.; Kortt, A. A.; Colman, P. M. Proteins 1992,14, 327) and Salmonella typhimurium (Crennell, S. J.; Garman, E. F.; Laver, W. G.; Vimr, E. R.; Taylor, G. L. Proc. Natl. Acad. Sci. U.S.A. 1993,90,9852) neuraminidases are very similar, yet the influenza enzyme works with retention of configuration (Chong, A. K.; Pegg, M. S.; Taylor, N. R.; von Itzstein, M. Eur. J. Biochem. 1992, 207, 335) and the S. typhimurium enzyme with inversion (Guo, X.; Sinnott, M. L. Biochem. J. 1993, 296, 291). To address the possibility that these two stereochemical outcomes may nonetheless be compatible with an essentially common sialosyl cation-stabilizing protein machinery, we have compared leaving group effects and (geometry-dependent) β-deuterium kinetic isotope effects for both enzymes. For the influenza enzyme, β1g values calculated on V and V/Kdiffer radically (-0.11 and -0.46, respectively), and we could detect neither β-deuterium nor leaving group 18O isotope effects on V for hydrolysis of the p-nitrophenyl glycoside at the optimal pH of 6, indicating, as previously found for the 4-methylumbelliferyl compound (Chong et al., 1992) that a step subsequent to glycon-aglycon cleavage determined V. Effects on V/K were not fully expressed at pH 6, in accord with the postulation of an isotope-insensitive step preceding bond cleavage. Intrinsic β-DKIEs on glycon-aglycon cleavage (measured as βD(V/K) at pH 9.5) of around 6% for the pro-R hydron and 8% for the pro-S are compatible with reaction through the B2,5 conformation of the sugar ring seen in the X-ray crystal structure of the neuraminidase-N-acetylneuraminic acid complex (Varghese et al., 1992). In accord with a single displacement mechanism for the S. typhimurium enzyme, the β1g values calculated on V and on V/K for the hydrolysis of seven aryl N-acetylneuraminides by this enzyme are both strongly negative (-0.53 and -0.80, respectively). β-DKIEs on V for the p-nitrophenyl compound are, however, around 60% of those on V/K, as is the leaving group 18O effect, when measured at optimal pH of 5.5. When measured at pH 8.0, the β-dideutero effect on V and on V/Kis the same, and the same as that on V/K at pH 5.5. Product release is therefore likely to partly govern V at optimum pH for this excellent (kcat = 7 X 103 s-1) substrate. The intrinsic leaving group 18O and individual pro-S and pro-R β-deuterium effects, all ~5%, coupled with the negative β1g values, indicate that the single chemical transition state for the S. typhimurium enzyme involves little proton donation to the leaving group, probably a sugar ring conformation approximating to ground-state 2C5, and substantial charge development at C2. The catalytic mechanisms of the two enzymes therefore differ radically.

Original languageEnglish (US)
Pages (from-to)5572-5578
Number of pages7
JournalJournal of the American Chemical Society
Volume116
Issue number13
DOIs
StatePublished - Jun 1 1994

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ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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