STEREOSELECTIVITY OF THE CHINESE-HAMSTER OVARY CELL SIALIDASE - SIALOSIDE HYDROLYSIS WITH OVERALL RETENTION OF CONFIGURATION

The stereochemical course of enzymatic hydrolysis by the soluble siali dase from Chinese hamster ovary cells, expressed as a recombinant prot ein 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 ca talysis was ascertained by monitoring the H3 axial and equatorial prot ons 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 th e reaction, arising from mutarotation of the alpha anomer, Similar stu dies were also carried out using the Salmonella typhimurium LT 2 siali dase, a protein of similar size and substrate specificity, Both enzyme s apparently cleave the alpha linked sialoside substrate with retentio n of configuration, Based on the observations of a wide variety of oth er glycohydrolytic enzymes that have shown a strong correlation of the stereoselectivity of catalysis,vith active site topology (Gebler et a t, J, Biol, Chem, 267, 12559-12561, 1992), the results obtained here s uggest that the microbial and mammalian sialidases have a homologous a ctive site architecture even though the molecules do not share signifi cant primary sequence similarities.