Stereoselectivity of the Chinese hamster ovary cell sialidase: sialoside hydrolysis with overall retention of configuration

'To whom correspondence should be addressed The stereochemical course of enzymatic hydrolysis by the soluble sialidase from Chinese hamster ovary cells, ex-pressed as a recombinant protein in insect Sf9 cells, was determined using proton nuclear magnetic resonance spec-troscopy. 4-Methyl umbelliferyl-N-acetyl neuraminic acid was employed as substrate, and the stereoselectivity of the enzyme catalysis was ascertained by monitoring the H3 axial and equatorial protons of the sialic acid product over the reaction course. At both high (3 U) and low concentra-tions (1 U) of the enzyme, the alpha anomer of the sialic acid was clearly observed as the initial reaction product. The corresponding beta anomer of sialic acid appeared much later in the reaction, arising from mutarotation of the alpha anomer. Similar studies were also carried out using the Salmonella typhimurium LT 2 sialidase, a protein of similar size and substrate specificity. Both enzymes appar-ently cleave the alpha linked sialoside substrate with reten-tion of configuration. Based on the observations of a wide variety of other glycohydrolytic enzymes that have shown a strong correlation of the stereoselectivity of catalysis with active site topology (Gebler et al, J. BioL Chem. 267, 12559-12561, 1992), the results obtained here suggest that the microbiaJ and mammalian sialidases have a homolo-gous active site architecture even though the molecules do not share significant primary sequence similarities. Key words: sialidase/NMR/enzyme mechanism/Chinese ham-ste