Kinetic and mechanistic characterization of the Sphingomyelinases D from Loxosceles intermedia spider venom

Envenomation by arachnids of the genus Loxosceles leads to local dermonecrosis and serious systemic toxicity mainly induced by sphingomyelinases D (SMase D). These enzymes catalyze the hydrolysis of sphingomyelin resulting in the formation of ceramide–phosphate and choline as well as the cleavage of...

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Published in:	Toxicon (Oxford) Vol. 47; no. 4; pp. 380 - 386
Main Authors:	Andrade, Sonia A. de, Murakami, Mario T., Cavalcante, Danielle P., Arni, Raghuvir K., Tambourgi, Denise V.
Format:	Journal Article
Language:	English
Published:	Oxford Elsevier Ltd 15-03-2006 Elsevier Science
Subjects:	Amino Acid Sequence Animal poisons toxicology. Antivenoms Binding Sites Biological and medical sciences Isoenzymes - chemistry Kinetic parameters Kinetics Loxosceles intermedia Loxosceles venoms Medical sciences Phosphoric Diester Hydrolases - chemistry Phosphoric Diester Hydrolases - metabolism Phosphoric Diester Hydrolases - toxicity Sphingomyelin Sphingomyelinase D Spider Venoms - chemistry Structure Structure-Activity Relationship Substrate Specificity Toxicology Sphingomyelin Sphingomyelinase D Loxosceles venoms Kinetic parameters Structure Molecular structure Animal origin Araneida Characterization Arachnida Arthropoda Venom Kinetics Kinetic parameter Invertebrata
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Summary:	Envenomation by arachnids of the genus Loxosceles leads to local dermonecrosis and serious systemic toxicity mainly induced by sphingomyelinases D (SMase D). These enzymes catalyze the hydrolysis of sphingomyelin resulting in the formation of ceramide–phosphate and choline as well as the cleavage of lysophosphatidyl choline generating the lipid mediator lysophosphatidic acid. We have, previously, cloned and expressed two functional SMase D isoforms, named P1 and P2, from Loxosceles intermedia venom and comparative protein sequence analysis revealed that they are highly homologous to SMase I from Loxosceles laeta which folds to form an (α/β) 8 barrel. In order to further characterize these proteins, pH dependence kinetic experiments and chemical modification of the two active SMases D isoforms were performed. We show here that the amino acids involved in catalysis and in the metal ion binding sites are strictly conserved in the SMase D isoforms from L. intermedia. However, the kinetic studies indicate that SMase P1 hydrolyzes sphingomyelin less efficiently than P2, which can be attributed to a substitution at position 203 (Pro–Leu) and local amino acid substitutions in the hydrophobic channel that could probably play a role in the substrate recognition and binding.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0041-0101 1879-3150
DOI:	10.1016/j.toxicon.2005.12.005