The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus spingomyelinase

Masahiro Tomita, Yasuji Ueda, Hiroomi Tamura, Ryo Taguchi, Hiroh Ikezawa

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol cause a drasic decrease in enzymatic activity, whereas the spingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the unchanged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards crythrocyte membranes.

Original languageEnglish
Pages (from-to)85-92
Number of pages8
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1203
Issue number1
DOIs
Publication statusPublished - 1993 Nov 10
Externally publishedYes

Fingerprint

Bacillus cereus
Acidic Amino Acids
Sphingomyelin Phosphodiesterase
Catalytic Domain
Dithiothreitol
Adsorption
Molecules
Membranes
Basic Amino Acids
Sphingomyelins
Erythrocyte Membrane
Disulfides
Amino Acids

Keywords

  • (B. cereus)
  • Amino acid residue, acidic
  • Chemical modification
  • Homologous sequence
  • Sequence analysis
  • Sphingomyelinase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

Cite this

The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus spingomyelinase. / Tomita, Masahiro; Ueda, Yasuji; Tamura, Hiroomi; Taguchi, Ryo; Ikezawa, Hiroh.

In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1203, No. 1, 10.11.1993, p. 85-92.

Research output: Contribution to journalArticle

@article{978788ca6b204a52809f5aebfbbf1002,
title = "The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus spingomyelinase",
abstract = "By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90{\%}. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol cause a drasic decrease in enzymatic activity, whereas the spingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the unchanged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50{\%}, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards crythrocyte membranes.",
keywords = "(B. cereus), Amino acid residue, acidic, Chemical modification, Homologous sequence, Sequence analysis, Sphingomyelinase",
author = "Masahiro Tomita and Yasuji Ueda and Hiroomi Tamura and Ryo Taguchi and Hiroh Ikezawa",
year = "1993",
month = "11",
day = "10",
doi = "10.1016/0167-4838(93)90039-T",
language = "English",
volume = "1203",
pages = "85--92",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus spingomyelinase

AU - Tomita, Masahiro

AU - Ueda, Yasuji

AU - Tamura, Hiroomi

AU - Taguchi, Ryo

AU - Ikezawa, Hiroh

PY - 1993/11/10

Y1 - 1993/11/10

N2 - By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol cause a drasic decrease in enzymatic activity, whereas the spingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the unchanged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards crythrocyte membranes.

AB - By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol cause a drasic decrease in enzymatic activity, whereas the spingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the unchanged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards crythrocyte membranes.

KW - (B. cereus)

KW - Amino acid residue, acidic

KW - Chemical modification

KW - Homologous sequence

KW - Sequence analysis

KW - Sphingomyelinase

UR - http://www.scopus.com/inward/record.url?scp=0027485151&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027485151&partnerID=8YFLogxK

U2 - 10.1016/0167-4838(93)90039-T

DO - 10.1016/0167-4838(93)90039-T

M3 - Article

VL - 1203

SP - 85

EP - 92

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 1

ER -