Stereoselectivity of the Chinese hamster ovary cell sialidase: sialoside hydrolysis with overall retention of configuration.
The stereochemical course of enzymatic hydrolysis by the soluble sialidase from Chinese hamster ovary cells, expressed as a recombinant protein in insect Sf9 cells, was determined using proton nuclear magnetic resonance spectroscopy. 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 concentrations (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 apparently cleave the alpha linked sialoside substrate with retention 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 microbial and mammalian sialidases have a homologous active site architecture even though the molecules do not share significant primary sequence similarities.
Kao
Y H
YH
Genentech, Inc., South San Francisco, CA 94080, USA.
Lerner
L
L
Warner
T G
TG
D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D006224:Cricetinae; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009439:Neuraminidase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D012794:Sialic Acids / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012794:Sialic Acids; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D009439:Neuraminidase