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
Xm. Guo et al., 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, Journal of the American Chemical Society, 116(13), 1994, pp. 5572-5578
The protein folds and presumed active site residues of influenza A (Va
rghese, J. N., McKimm-Breschkin, J. L.; Caldwell, J. B.; Kortt, A; A.;
Colman, P. M. Proteins 1992, 14, 327) and Salmonella typhimurium (Cre
nnell, 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 configuratio
n (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 p
ossibility that these two stereochemical outcomes may nonetheless be c
ompatible with an essentially common sialosyl cation-stabilizing prote
in machinery, we have compared leaving group effects and (geometry-dep
endent) beta-deuterium kinetic isotope effects for both enzymes. For t
he influenza enzyme, Be values calculated on V and V/K differ radicall
y (-0.11 and -0.46, respectively), and we could detect neither beta-de
uterium nor leaving group O-18 isotope effects on V for hydrolysis of
the p-nitrophenyl glycoside at the optimal pH of 6, indicating, as pre
viously found for the 4-methylumbelliferyl compound (Chong et al., 199
2) that a step subsequent to glycon-aglycon cleavage determined V. Eff
ects on V/K were not fully expressed at pH 6, in accord with the postu
lation of an isotope-insensitive step preceding bond cleavage. Intrins
ic 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 co
mpatible with reaction through the B-2,B-5 conformation of the sugar r
ing seen in the X-ray crystal structure of the neuraminidase-N-acetyln
euraminic acid complex (Varghese et al., 1992). In accord with a singl
e displacement mechanism for the S. typhimurium enzyme, the beta(lg) v
alues calculated on V and on V/K for the hydrolysis of seven aryl N-ac
etylneuraminides by this enzyme are both strongly negative (-0.53 and
-0.80, respectively). beta-DKIEs on V for the p-nitrophenyl compound a
re, 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, t
he 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 g
overn Vat optimum pH for this excellent (k(cat) = 7 X 10(3) s(-1)) sub
strate. 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 f
or the S. typhimurium enzyme involves little proton donation to the le
aving group, probably a sugar ring conformation approximating to groun
d-state C-2(5), and substantial charge development at C2. The catalyti
c mechanisms of the two enzymes therefore differ radically.