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 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 (alpha/beta)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.
de Andrade
Sonia A
SA
Laboratório de Imunoquímica, Instituto Butantan, Av. Prof. Vital Brazil, 1500, CEP 05508-900, São Paulo, SP 05503-900, Brazil.
Murakami
Mario T
MT
Cavalcante
Danielle P
DP
Arni
Raghuvir K
RK
Tambourgi
Denise V
DV
D000595:Amino Acid Sequence; D001665:Binding Sites; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D013111:Spider Venoms / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007527:Isoenzymes; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; C055160:sphingomyelin phosphodiesterase D