A kinetic-isotope-effect study of catalysis by Vibrio cholerae neuraminidase.

Michaelis-Menten parameters for hydrolysis of seven aryl N-acetyl alpha-D-neuraminides by Vibrio cholerae neuraminidase at pH 5.0 correlate well with the leaving-group pKa (delta pK 3.0; beta 1g (V/K) = -0.73, r = -0.93; beta 1g (V) = -0.25; r = -0.95). The beta-deuterium kinetic-isotope effect, beta D2(V), for the p-nitrophenyl glycoside is the same at the optimum pH of 5.0 (1.059 +/- 0.010) as at pH 8.0 (1.053 +/- 0.010), suggesting that isotope effects are fully expressed with this substrate at the optimum pH. For this substrate at pH 5.0, leaving group 18O effects are 18(V) = 1.040 +/- 0.016 and 18(V/K) = 1.046 +/- 0.015, and individual secondary deuterium effects are beta proRD(V) = 1.037 +/- 0.014, beta proSD(V) = 1.018 +/- 0.015, beta proRD(V/K) = 1.030 +/- 0.017, beta proSD(V/K) = 1.030 +/- 0.017. All isotope effects, and the beta 1g(V/K) value are in accord with the first chemical step being both the first irreversible and the rate-determining step in enzyme turnover, with a transition state in which there is little proton donation to the leaving group, the C-O bond is largely cleaved, there is significant nucleophilic participation, and the sugar ring is in a conformation derived from the ground-state 2C5 chair. The apparent conflict between the beta 1g (V) value of -0.25 with all the kinetic-isotope-effect data can be resolved by the postulation of an interaction between the pi system of the aglycone ring and an anionic or nucleophilic group on the enzyme.