Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases

Abstract

BackgroundsStreptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate.ResultsIn this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p-NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses.ConclusionWe developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.

Streptococcus pneumoniae NanC

“…For A, the S. pneumoniae TIGR4 nanC gene was expressed in the pet21b vector ( 28 ). The construct encompassed residues 28–740.…”

“…NanB is an intramolecular trans -sialidase generating 2,7-anhydro-Neu5Ac as a reaction product ( 24 , 25 ). Despite the high sequence similarity to NanB, NanC has been shown to differ in that it initially generates Neu5Ac2en, a molecule that inhibits NanA with a K i of 2 μ m ( 24 , 28 ). In fact, Neu5Ac2en appears to be a nonspecific inhibitor of hydrolytic sialidases with micromolar inhibition also observed against influenza virus neuraminidase ( 29 ), the human sialidases ( 30 , 31 ), and VcNA from Vibrio cholerae ( 32 ).…”

“…In fact, Neu5Ac2en appears to be a nonspecific inhibitor of hydrolytic sialidases with micromolar inhibition also observed against influenza virus neuraminidase ( 29 ), the human sialidases ( 30 , 31 ), and VcNA from Vibrio cholerae ( 32 ). Neu5Ac2en is a very poor inhibitor of NanC with a K i of 2–3 m m ( 27 , 28 ). Following depletion of the α2–3-linked substrate, NanC can hydrate Neu5Ac2en to Neu5Ac ( 27 ).…”

Enhancement of bioconversion gangliosides to monosialotetrahexosylganglioside by in situ sialic acid removal and recovery

“…The K m value was obtained by the slope and longitudinal intercept of the line. The inhibitory constant ( K i ) was obtained by plotting K * m /K m on inhibitor concentration, using a competitive inhibition model, where K m */ K m = 1 + [I]/ K i . The value with an asterisk was expressed as the value measured in the system in which the inhibitor was added.…”

Recognition of diazirine-modified O-GlcNAc by human O-GlcNAcase

“…27,34–36 Indeed Hayre et al demonstrated accurate determination of kinetics of sialidase-catalyzed reactions using a p -nitrophenol reporter and continuous absorbance measurements at pH 6.0. 34 Although the reaction buffer was at a pH lower than the pK a of 4-nitrophenol, a substantial increase in absorbance was detectable when OGA activity was present. Changes in absorbance at 405 nm were linear with respect to time over the period between 5 and 35 minutes (Figs.…”