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

摘要: The sequence similarity with bacterial neutral sphingomyelinase resulted in the isolation of putative mammalian counterparts and, subsequently, identification similar molecules a number other eukaryotic organisms. Based on similarities and previous characterization enzymes, we have chemically modified specific residues performed site-directed mutagenesis order to identify critical catalytic determinants for membrane localization. Modification histidine substrate protection experiments demonstrated presence reactive within active site. Site directed suggested an essential role catalysis two (His-136 His-272), which are conserved all sequences. Mutations additional histidines (His-138 His-151), only eukaryotes, reduced activity. In addition sphingomyelin, enzyme also hydrolyzed lysophosphatidylcholine. Exposure oxidizing environment or modification cysteine using several compounds inactivated enzyme. Site-directed eight gel-shift analysis that these did not participate reaction involvement cysteines formation/breakage disulfide bonds, could underlie reversible inactivation by compounds. Cellular localization studies series deletion mutants, expressed as green fluorescent protein fusion proteins, transmembrane region contains endoplasmic reticulum