title: Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases.
abstract: BACKGROUND
  Streptococcus 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Dipartimento di Biotecnologie, Università degli Studi di Siena, I-53100, Siena, Italy.
authors.forename: Jasvinder Kaur
authors.initials: JK
authors.lastname: Hayre
authors.forename: Guogang
authors.initials: G
authors.lastname: Xu
authors.forename: Luisa
authors.initials: L
authors.lastname: Borgianni
authors.forename: Garry L
authors.initials: GL
authors.lastname: Taylor
authors.forename: Peter W
authors.initials: PW
authors.lastname: Andrew
authors.forename: Jean-Denis
authors.initials: JD
authors.lastname: Docquier
authors.forename: Marco R
authors.initials: MR
authors.lastname: Oggioni
mesh_terms: D055162:Biocatalysis; D002021:Buffers; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013296:Streptococcus pneumoniae / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D002021:Buffers; D011994:Recombinant Proteins; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid