Substrate and product specificity of Arthrobacter sialophilus neuraminidase

Arthrobacter sialophilus neuraminidase catalyzes the hydrolysis of N-acetylneuraminyl-alpha-oxygen, nitrogen, and azido glycosides. The most effective of those substrates examined was N-acetylneuraminyl-alpha-4-methylumbelliferylglycoside (AcNeu-alpha-4-MU; Km app, 0.0193 mM; kcat, 136.4 sec-1). The products resulting from the enzymic hydrolysis of N-acetylneuraminyl-alpha-azido-glycoside were N-acetylneuraminic acid and azide ion. N-acetylneuraminyl-alpha-2,3-thiogalactylglycoside and N-acetylneuraminyl-alpha-2,6-thiogalactylglycoside were competitive inhibitors of the enzyme having KI values of 1.52 mM and 1.70 mM, respectively. Dissociation constants for these thioglycosides were also determined by fluorescence enzyme titrations which gave values similar to those determined kinetically. N-Acetylneuraminic acid, but not its methyl ester, was a competitive inhibitor of neuraminidase. Its KI value, 0.18 mM, was also determined by both methods. 5-Acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-talo-nonulosonic acid (2-deoxy-4-epi-AcNeu) was found to be a weak competitive inhibitor (KI, 12.1 mM). A. sialophilus neuraminidase further catalyzes transglycosidation reactions with methanol as acceptor. Methanol had no effect on the release of 4-MU by enzymatic hydrolysis of AcNeu-alpha-4-MU, suggesting that the formation of the enzyme-glycone intermediate is the rate-determining step. The anomeric configuration of the product of this reaction, as shown by 13C-nmr spectroscopy, is N-acetylneuraminyl-alpha-methylglycoside. Neuraminidase, therefore, catalyzes its reactions with overall retention of configuration.