His151 and His296 are the acid-base catalytic residues of Bacillus cereus sphingomyelinase in sphingomyelin hydrolysis.
Bacillus cereus sphingomyelinase belongs to the Mg(2+)-dependent neutral sphingomyelinase, which hydrolyses sphingomyelin to phosphocholine and ceramide, and acts as an extracellular hemolysin. The triplet residues, His151-Asp195-His296, of the enzyme are highly conserved among bacterial and mammalian Mg(2+)-dependent neutral sphingomyelinases. The triplet residues converge on the active-site pocket of the 3D model of the enzyme. To investigate the function of these residues in the acid-base catalysis, we introduced several mutations for each residue by site-directed mutagenesis. Hemolytic and hydrolytic activities of the enzyme, abolished by the mutations at Asp195 and His296, revealed that these residues are critical for the catalytic function. The effect of the divalent metal cations on the pH dependency of the hydrolytic activities indicates that His296 corresponds to the most acidic ionizable group as a general base. The mutagenesis at His151 was also deleterious; however, the H151A and H151Q mutant enzymes partially retained their activities. The H151A mutation affected the most basic ionizable group, suggesting that His151 may act as a general acid in catalysis. By the structural basis of the 3D model, Asp195 must maintain not only the appropriate spatial arrangement but also pK(a)s of His151 and His296. Taking into consideration all of these, we proposed the acid-base catalytic mechanism of B. cereus sphingomyelinase.
Obama
Takashi
T
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan.
Fujii
Shinobu
S
Ikezawa
Hiroh
H
Ikeda
Kiyoshi
K
Imagawa
Masayoshi
M
Tsukamoto
Kikuo
K
D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D017354:Point Mutation; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism*; D013109:Sphingomyelins / Q000235:genetics / Q000378:metabolism*
D013109:Sphingomyelins; D006639:Histidine; D013108:Sphingomyelin Phosphodiesterase