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, Cys(10), 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. Cys(501), 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 Cys(501) 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.
Murphy
Deirdre M
DM
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575, USA.
Ivanenkov
Vasily V
VV
Kirley
Terence L
TL
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001921:Brain / Q000201:enzymology; D003432:Cross-Linking Reagents; D003545:Cysteine; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010726:Phosphodiesterase Inhibitors; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D002731:4-Chloromercuribenzenesulfonate; D011755:Pyrophosphatases; C439652:nucleoside-triphosphate diphosphohydrolase 3; D003545:Cysteine