title: Mechanistic requirements for the efficient enzyme-catalyzed hydrolysis of thiosialosides.
abstract: The sialidase from Micromonospora viridifaciens has been found to catalyze the hydrolysis of aryl 2-thio-alpha-D-sialosides with remarkable efficiency: the first- and second-order rate constants, kcat and kcat/Km, for the enzyme-catalyzed hydrolysis of PNP-S-NeuAc are 196 +/- 5 s(-1) and (6.7 +/- 0.7) x 10(5) M(-1) s(-1), respectively. A reagent panel of eight aryl 2-thio-alpha-D-sialosides was synthesized and used to probe the mechanism for the M. viridifaciens sialidase-catalyzed hydrolysis reaction. In the case of the wild-type enzyme, the derived Brønsted parameters (beta(lg)) on kcat and kcat/Km are -0.83 +/- 0.11 and -1.27 +/- 0.17 for substrates with thiophenoxide leaving groups of pKa values > or = 4.5. For the general-acid mutant, D92G, the derived beta(lg) value on kcat for the same set of leaving groups is -0.82 +/- 0.12. When the conjugate acid of the departing thiophenol was < or = 4.5, the derived Brønsted slopes for both the wild-type and the D92G mutant sialidase were close to zero. In contrast, the nucleophilic mutant, Y370G, did not display a similar break in the Brønsted plots, and the corresponding values for beta(lg), for the three most reactive aryl 2-thiosialosides, on kcat and kcat/Km are -0.76 +/- 0.28 and -0.84 +/- 0.04, respectively. Thus, for the Y370G enzyme glycosidic C-S bond cleavage is rate-determining for both kcat and kcat/Km, whereas, for both the wild-type and D92G mutant enzymes, the presented data are consistent with a change in rate-determining step from glycosidic C-S bond cleavage for substrates in which the pKa of the conjugate acid of the leaving group is > or = 4.5, to either deglycosylation (kcat) or a conformational change that occurs prior to C-S bond cleavage (kcat/Km) for the most activated leaving groups. Thus, the enzyme-catalyzed hydrolysis of 2-thiosialosides is strongly catalyzed by the nucleophilic tyrosine residue, yet the C-S bond cleavage does not require the conserved aspartic acid residue (D92) to act as a general-acid catalyst.
authors.affiliation: Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Arun A
authors.initials: AA
authors.lastname: Narine
authors.forename: Jacqueline N
authors.initials: JN
authors.lastname: Watson
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bennet
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D006868:Hydrolysis; D008847:Micromonospora / Q000201:enzymology; D009439:Neuraminidase / Q000737:chemistry / Q000378:metabolism*; D012794:Sialic Acids / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D012794:Sialic Acids; D009439:Neuraminidase