Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities - Analysis by chemical modification and site-directed mutagenesis

The sequence similarity with bacterial neutral sphingomyelinase resulted in the isolation of putative mammalian counterparts and, subsequently, identi fication of similar molecules in a number of other eukaryotic organisms. Ba sed on sequence similarities and previous characterization of the mammalian enzymes, we have chemically modified specific residues and pet-formed site -directed mutagenesis in order to identify critical catalytic residues and determinants for membrane localization, Modification of histidine residues and the substrate protection experiments demonstrated the presence of react ive histidine residues within the active site. Site directed mutagenesis su ggested an essential role in catalysis for two histidine residues (His-136 and His-272), which are conserved in all sequences. Mutations of two additi onal histidines (His-138 and His-151), consented only in eukaryotes, result ed in reduced neutral sphingomyelinase activity. In addition to sphingomyel in, the enzyme also hydrolyzed lysophosphatidylcholine, Exposure to an oxid izing environment or modification of cysteine residues using several specif ic compounds also inactivated the enzyme. Site-directed mutagenesis of eigh t cysteine residues and gel-shift analysis demonstrated that these residues did not participate in the catalytic reaction and suggested the involvemen t of cysteines in the formation/breakage of disulfide bonds, which could un derlie the reversible inactivation by the oxidizing compounds. Cellular loc alization studies of a series of deletion mutants, expressed as green fluor escent protein fusion proteins, demonstrated that the transmembrane region contains determinants for the endoplasmic reticulum localization.