CATALYSIS BY 2 SIALIDASES WITH THE SAME PROTEIN FOLD BUT DIFFERENT STEREOCHEMICAL COURSES - A MECHANISTIC COMPARISON OF THE ENZYMES FROM INFLUENZA-A VIRUS AND SALMONELLA-TYPHIMURIUM

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.; Carman, 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) beta-deuterium kinetic isotope effects for both enzymes. For the influenza enzyme, Be values calculated on V and V/K differ radically (-0.11 and -0.46, respectively), and we could detect neither beta-deuterium nor leaving group O-18 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 beta-DKIEs on glycon-aglycon cleavage (measured as (beta D)(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 B-2,B-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 beta(lg) 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). beta-DKIEs on V for the p-nitrophenyl compound are, however, around 60% of those on V/K, as is the leaving group O-18 effect, when measured at optimal pH of 5.5. When measured at pH 8.0, the beta-dideutero effect on V and on V/K is the same, and the same as that on V/K at pH 5.5. Product release is therefore likely to partly govern Vat optimum pH for this excellent (k(cat) = 7 X 10(3) s(-1)) substrate. The intrinsic leaving group O-18 and individual pro-S and pro-R beta-deuterium effects, all similar to 5%, coupled with the negative beta(lg) 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 C-2(5), and substantial charge development at C2. The catalytic mechanisms of the two enzymes therefore differ radically.