Binding of ATP and its derivatives to selenophosphate synthetase from Escherichia coli

Y. V. Preabrazhenskaya, Ick Young Kim, T. C. Stadtman

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Mechanistically similar selenophosphate synthetases (SPS) have been isolated from different organisms. SPS from Escherichia coli is an ATP-dependent enzyme with a C-terminal glycine-rich Walker sequence that has been assumed to take part in the first step of ATP binding. Three C-terminally truncated mutants of SPS, containing the N-terminal 238 (SPS238), 262 (SPS 262), and 332 (SPS332) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS 262 and SPS332) have been purified to homogeneity. SPS238 has been obtained in a highly purified form. Binding of the fluorescent ATP-derivative TNP-ATP and Mn-ATP to the proteins was examined for all truncated mutants of SPS and a catalytically inactive C17S mutant. It has been shown that TNP-ATP can be used as a structural probe for ATP-binding sites of SPS. We observed two TNP-ATP binding sites per molecule of enzyme for wild-type SPS and SPS332 mutant and one TNP-ATP binding site for SPS238 mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [14C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS332, SPS262, and SPS238 were 2, 1.6, and 1, respectively. The C17S mutant exhibits about one third of wild type SPS TNP-ATP-binding ability and converts 12% of ATP in the ATPase reaction to ADP in the absence of selenide. The C-terminus contributes two thirds to the TNP-ATP binding; SPS238 likely has one ATP-binding site removed by truncation.

Original languageEnglish
Pages (from-to)910-916
Number of pages7
JournalBiochemistry (Moscow)
Volume74
Issue number8
DOIs
Publication statusPublished - 2009 Aug 1
Externally publishedYes

Fingerprint

Escherichia coli
Adenosine Triphosphate
Derivatives
Binding Sites
Stoichiometry
selenophosphate synthetase
Amino Acids
Column chromatography
Proteins
Enzymes
Glycine
Adenosine Diphosphate
Gel Chromatography
Adenosine Triphosphatases
2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate
Gels
High Pressure Liquid Chromatography
Molecules

Keywords

  • ATP-binding
  • Fluorescence enhancement
  • Selenophosphate synthetase
  • Truncated mutants

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

Cite this

Binding of ATP and its derivatives to selenophosphate synthetase from Escherichia coli. / Preabrazhenskaya, Y. V.; Kim, Ick Young; Stadtman, T. C.

In: Biochemistry (Moscow), Vol. 74, No. 8, 01.08.2009, p. 910-916.

Research output: Contribution to journalArticle

Preabrazhenskaya, Y. V. ; Kim, Ick Young ; Stadtman, T. C. / Binding of ATP and its derivatives to selenophosphate synthetase from Escherichia coli. In: Biochemistry (Moscow). 2009 ; Vol. 74, No. 8. pp. 910-916.
@article{eaff8400198c41159f09c3b7d57edfa2,
title = "Binding of ATP and its derivatives to selenophosphate synthetase from Escherichia coli",
abstract = "Mechanistically similar selenophosphate synthetases (SPS) have been isolated from different organisms. SPS from Escherichia coli is an ATP-dependent enzyme with a C-terminal glycine-rich Walker sequence that has been assumed to take part in the first step of ATP binding. Three C-terminally truncated mutants of SPS, containing the N-terminal 238 (SPS238), 262 (SPS 262), and 332 (SPS332) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS 262 and SPS332) have been purified to homogeneity. SPS238 has been obtained in a highly purified form. Binding of the fluorescent ATP-derivative TNP-ATP and Mn-ATP to the proteins was examined for all truncated mutants of SPS and a catalytically inactive C17S mutant. It has been shown that TNP-ATP can be used as a structural probe for ATP-binding sites of SPS. We observed two TNP-ATP binding sites per molecule of enzyme for wild-type SPS and SPS332 mutant and one TNP-ATP binding site for SPS238 mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [14C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS332, SPS262, and SPS238 were 2, 1.6, and 1, respectively. The C17S mutant exhibits about one third of wild type SPS TNP-ATP-binding ability and converts 12{\%} of ATP in the ATPase reaction to ADP in the absence of selenide. The C-terminus contributes two thirds to the TNP-ATP binding; SPS238 likely has one ATP-binding site removed by truncation.",
keywords = "ATP-binding, Fluorescence enhancement, Selenophosphate synthetase, Truncated mutants",
author = "Preabrazhenskaya, {Y. V.} and Kim, {Ick Young} and Stadtman, {T. C.}",
year = "2009",
month = "8",
day = "1",
doi = "10.1134/S0006297909080136",
language = "English",
volume = "74",
pages = "910--916",
journal = "Biochemistry. Biokhimiia",
issn = "0006-2979",
publisher = "Maik Nauka-Interperiodica Publishing",
number = "8",

}

TY - JOUR

T1 - Binding of ATP and its derivatives to selenophosphate synthetase from Escherichia coli

AU - Preabrazhenskaya, Y. V.

AU - Kim, Ick Young

AU - Stadtman, T. C.

PY - 2009/8/1

Y1 - 2009/8/1

N2 - Mechanistically similar selenophosphate synthetases (SPS) have been isolated from different organisms. SPS from Escherichia coli is an ATP-dependent enzyme with a C-terminal glycine-rich Walker sequence that has been assumed to take part in the first step of ATP binding. Three C-terminally truncated mutants of SPS, containing the N-terminal 238 (SPS238), 262 (SPS 262), and 332 (SPS332) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS 262 and SPS332) have been purified to homogeneity. SPS238 has been obtained in a highly purified form. Binding of the fluorescent ATP-derivative TNP-ATP and Mn-ATP to the proteins was examined for all truncated mutants of SPS and a catalytically inactive C17S mutant. It has been shown that TNP-ATP can be used as a structural probe for ATP-binding sites of SPS. We observed two TNP-ATP binding sites per molecule of enzyme for wild-type SPS and SPS332 mutant and one TNP-ATP binding site for SPS238 mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [14C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS332, SPS262, and SPS238 were 2, 1.6, and 1, respectively. The C17S mutant exhibits about one third of wild type SPS TNP-ATP-binding ability and converts 12% of ATP in the ATPase reaction to ADP in the absence of selenide. The C-terminus contributes two thirds to the TNP-ATP binding; SPS238 likely has one ATP-binding site removed by truncation.

AB - Mechanistically similar selenophosphate synthetases (SPS) have been isolated from different organisms. SPS from Escherichia coli is an ATP-dependent enzyme with a C-terminal glycine-rich Walker sequence that has been assumed to take part in the first step of ATP binding. Three C-terminally truncated mutants of SPS, containing the N-terminal 238 (SPS238), 262 (SPS 262), and 332 (SPS332) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS 262 and SPS332) have been purified to homogeneity. SPS238 has been obtained in a highly purified form. Binding of the fluorescent ATP-derivative TNP-ATP and Mn-ATP to the proteins was examined for all truncated mutants of SPS and a catalytically inactive C17S mutant. It has been shown that TNP-ATP can be used as a structural probe for ATP-binding sites of SPS. We observed two TNP-ATP binding sites per molecule of enzyme for wild-type SPS and SPS332 mutant and one TNP-ATP binding site for SPS238 mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [14C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS332, SPS262, and SPS238 were 2, 1.6, and 1, respectively. The C17S mutant exhibits about one third of wild type SPS TNP-ATP-binding ability and converts 12% of ATP in the ATPase reaction to ADP in the absence of selenide. The C-terminus contributes two thirds to the TNP-ATP binding; SPS238 likely has one ATP-binding site removed by truncation.

KW - ATP-binding

KW - Fluorescence enhancement

KW - Selenophosphate synthetase

KW - Truncated mutants

UR - http://www.scopus.com/inward/record.url?scp=69249111622&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=69249111622&partnerID=8YFLogxK

U2 - 10.1134/S0006297909080136

DO - 10.1134/S0006297909080136

M3 - Article

VL - 74

SP - 910

EP - 916

JO - Biochemistry. Biokhimiia

JF - Biochemistry. Biokhimiia

SN - 0006-2979

IS - 8

ER -