Identification of Cysteine Residues Responsible for Oxidative Cross-linking and Chemical Inhibition of Human Nucleoside-triphosphate Diphosphohydrolase 3*

Cysteine-to-serine mutations were constructed to test the functional and structural significance of the three non-extracellular cysteine residues in ecto-nucleoside-triphosphate diphosphohydrolase 3 (eNTPDase3). None of these cysteines were found to be essential for enzyme activity. However, Cys10, located on the short N-terminal cytoplasmic tail, was found to be responsible for dimer formation occurring via oxidation during membrane preparation as well as for dimer cross-linking resulting from exogenously added sulfhydryl-specific cross-linking agents. The resistance to further cross-linking of these dimers into higher order oligomers by lysine-specific cross-linkers suggests that this enzyme may form its native tetrameric structure as a “dimer of dimers” with nonequivalent interactions between subunits. Cys501, located in the hydrophobic C-terminal membrane-spanning domain of eNTPDase3, was found to be the site of chemical modification by a sulfhydryl-specific reagent, p-chloromercuriphenylsulfonic acid (pCMPS), leading to inhibition of enzyme activity. The effect of pCMPS was negligible after dissociation of the enzyme into monomers by Triton X-100, suggesting that the mechanism of inhibition is dependent on the oligomeric structure. Because Cys501 is accessible for modification by the membrane-impermeant reagent pCMPS, we hypothesize that eNTPDase3 (and possibly other eNTPDases) contains a water-filled crevice allowing access of water and hydrophilic compounds to at least part of the protein's C-terminal membrane-spanning helix.

• Publication year

  2002

• Venue

  Journal of Biological Chemistry

• Publication date

  2002-02-22

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1074/JBC.M110105200PMID 11748229
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• The importance of histidine residues in human ecto-nucleoside triphosphate diphosphohydrolase-3 as determined by site-directed mutagenesis.

  2001cited by this paper
• Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of conserved glycine residues and the identification of additional conserved protein motifs in eNTPDases.

  2001cited by this paper
• Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli.

  2001cited by this paper
• Regulation of chicken gizzard ecto-ATPase activity by modulators that affect its oligomerization status.

  2001cited by this paper
• Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of residues in the apyrase conserved regions.

  2001cited by this paper
• The presence of an aqueous cavity in the proton-pumping pathway of the pyridine nucleotide transhydrogenase of Escherichia coli is suggested by the reaction of the enzyme with sulfhydryl inhibitors.

  2000cited by this paper
• Expression and Characterization of Human Ecto‐ATPase and Chimeras with CD39 Ecto‐Apyrase

  2000cited by this paper
• Benzodiazepines Induce a Conformational Change in the Region of the γ-Aminobutyric Acid Type A Receptor α1-Subunit M3 Membrane-Spanning Segment

  2000cited by this paper
• Benzodiazepines induce a conformational change in the region of the gamma-aminobutyric acid type A receptor alpha(1)-subunit M3 membrane-spanning segment.

  2000cited by this paper
• Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family.

  2000cited by this paper
• Expression and Characterization of Soluble and Membrane-bound Human Nucleoside Triphosphate Diphosphohydrolase 6 (CD39L2)*

  2000cited by this paper
• Cysteine-scanning mutagenesis of transmembrane segment 11 of the GLUT1 facilitative glucose transporter.

  2000cited by this paper
• Biochemical characterization of CD39L4.

  2000cited by this paper
• Palmitoylation Targets CD39/Endothelial ATP Diphosphohydrolase to Caveolae*

  2000cited by this paper
• CD39L2, a gene encoding a human nucleoside diphosphatase, predominantly expressed in the heart.

  2000cited by this paper
• Site-directed mutagenesis of a human brain ecto-apyrase: evidence that the E-type ATPases are related to the actin/heat shock 70/sugar kinase superfamily.

  1999cited by this paper
• Glycosylation is essential for functional expression of a human brain ecto-apyrase.

  1999cited by this paper
• Ectonucleotidases of avian gizzard smooth muscle and liver plasma membranes: a comparative study.

  1999cited by this paper
• Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase.

  1999cited by this paper
• The CD39-like gene family: identification of three new human members (CD39L2, CD39L3, and CD39L4), their murine homologues, and a member of the gene family from Drosophila melanogaster.

  1998cited by this paper
• The Transmembrane Domains of Ectoapyrase (CD39) Affect Its Enzymatic Activity and Quaternary Structure*

  1998cited by this paper
• Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPases and CD39 ecto-apyrases1.

  1998cited by this paper
• Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPases and CD39 ecto-apyrases

  1998cited by this paper
• Histidine 225, a Residue of the NhaA-Na+/H+ Antiporter of Escherichia coli Is Exposed and Faces the Cell Exterior*

  1997cited by this paper
• Control of cell membrane ecto-ATPase by oligomerization state: intermolecular cross-linking modulates ATPase activity.

  1996cited by this paper
• Residues in the pathway through a membrane transporter.

  1995cited by this paper
• Ecto-ATPases: identities and functions.

  1995cited by this paper
• Prevalence of the mercurial-sensitive EctoATPase in human small cell lung carcinoma: characterization and partial purification.

  1994cited by this paper
• Increased uniformity in the response of the coomassie blue G protein assay to different proteins.

  1990cited by this paper
• Purification and characterization of the Mg2+-ATPase from rabbit skeletal muscle transverse tubule.

  1988cited by this paper
• A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay.

  1988cited by this paper
• Protein assay sensitive at nanogram levels.

  1987cited by this paper
• THE COLORIMETRIC DETERMINATION OF PHOSPHORUS

  1925cited by this paper

• The Transmembrane Glutamate Serves as a pH Sensor for Tha4 Oligomerization During Twin Arginine Transport of Proteins

  2025cites this paper
• Targeted Covalent Inhibitors Allosterically Deactivate the DEDDh Lassa Fever Virus NP Exonuclease from Alternative Distal Sites

  2021cites this paper
• Cellular function and molecular structure of ecto-nucleotidases

  2012cites this paper
• The GDA1_CD39 superfamily: NTPDases with diverse functions

  2011cites this paper
• Proline residues link the active site to transmembrane domain movements in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)

  2010cites this paper
• Epitope mapping in cell surface proteins by site-directed masking: defining the structural elements of NTPDase3 inhibition by a monoclonal antibody.

  2010cites this paper
• The single NTPase gene of Drosophila melanogaster encodes an intracellular nucleoside triphosphate diphosphohydrolase 6 (NTPDase6).

  2009cites this paper
• Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase‐3

  2009cites this paper
• Conserved polar residues stabilize transmembrane domains and promote oligomerization in human nucleoside triphosphate diphosphohydrolase 3.

  2009cites this paper
• Structure function studies on lectin nucleotide phosphohydrolases (LNPs).

  2008cites this paper
• Inhibition of human NTPDase 2 by modification of an intramembrane cysteine by p-chloromercuriphenylsulfonate and oxidative cross-linking of the transmembrane domains.

  2008cites this paper
• Transmembrane domain interactions affect the stability of the extracellular domain of the human NTPDase 2.

  2008cites this paper
• Trafficking and intracellular ATPase activity of human ecto-nucleotidase NTPDase3 and the effect of ER-targeted NTPDase3 on protein folding.

  2008cites this paper
• Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: implications for modulation of multiple homeostatic systems including feeding and sleep-wake behaviors.

  2006cites this paper
• The structure of the nucleoside triphosphate diphosphohydrolases (NTPDases) as revealed by mutagenic and computational modeling analyses

  2006influential citation
• Noise-induced up-regulation of NTPDase3 expression in the rat cochlea: Implications for auditory transmission and cochlear protection.

  2006cites this paper
• Calcium-dependent Dimerization of Human Soluble Calcium Activated Nucleotidase

  2006cites this paper
• Cloning and characterization of the ecto-nucleotidase NTPDase3 from rat brain: Predicted secondary structure and relation to other members of the E-NTPDase family and actin

  2005cites this paper
• Characterization of disulfide bonds in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): implications for NTPDase structural modeling.

  2005cites this paper
• Electroactivity of Proteins: Possibilities in Biomedicine and Proteomics

  2005cites this paper
• Identification of a tyrosine residue responsible for N-acetylimidazole-induced increase of activity of ecto-nucleoside triphosphate diphosphohydrolase 3

  2005cites this paper
• Derivatisation of peptides with osmium tetroxide, 2,2′-bipyridine: capillary electrophoretic and MALDI–TOF mass spectrometric study

  2004cites this paper
• Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)

  2004cites this paper
• Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3.

  2004cites this paper
• Determination of native oligomeric state and substrate specificity of rat NTPDase1 and NTPDase2 after heterologous expression in Xenopus oocytes.

  2003cites this paper
• Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes.

  2003cites this paper
• Bacterial expression, characterization, and disulfide bond determination of soluble human NTPDase6 (CD39L2) nucleotidase: implications for structure and function.

  2003cites this paper
• Identificação das características cinéticas, expressão e localização das ecto-nucleotidases em cultura de astrócitos e linhagens de gliomas

  2003cites this paper
• Asparagine 81, an invariant glycosylation site near apyrase conserved region 1, is essential for full enzymatic activity of ecto-nucleoside triphosphate diphosphohydrolase 3.

  2003cites this paper
• Cloning, expression, and characterization of a soluble calcium-activated nucleotidase, a human enzyme belonging to a new family of extracellular nucleotidases.

  2002cites this paper