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Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)

Cell membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are homooligomeric, with native quaternary structure required for maximal enzyme activity. In this study, we mutated lysine 79 in human ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3). The residue correspond...

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Autores principales: Basu, Saswata, Murphy-Piedmonte, Deirdre M., Kirley, Terence L.
Formato: Texto
Lenguaje:English
Publicado: Springer Netherlands 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2096571/
https://www.ncbi.nlm.nih.gov/pubmed/18404400
http://dx.doi.org/10.1007/s11302-004-4741-8
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author Basu, Saswata
Murphy-Piedmonte, Deirdre M.
Kirley, Terence L.
author_facet Basu, Saswata
Murphy-Piedmonte, Deirdre M.
Kirley, Terence L.
author_sort Basu, Saswata
collection PubMed
description Cell membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are homooligomeric, with native quaternary structure required for maximal enzyme activity. In this study, we mutated lysine 79 in human ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3). The residue corresponding to lysine 79 in NTPDase3 is conserved in all known cell surface membrane NTPDases (NTPDase1, 2, 3, and 8), but not in the soluble, monomeric NTPDases (NTPDase5 and 6), or in the intracellular, two transmembrane NTPDases (NTPDase4 and 7). This conserved lysine is located between apyrase conserved region 1 (ACR1) and an invariant glycosylation site (N81), in a region previously hypothesized to be important for NTPDase3 oligomeric structure. This lysine residue was mutated to several different amino acids, and all mutants displayed substantially decreased nucleotidase activities. A basic amino acid at this position was found to be important for the increase of nucleotidase activity observed after treatment with the lectin, concanavalin A. After solubilization with Triton X-100, mutants showed little or no decrease in activity, unlike the wild-type enzyme, suggesting that the lysine at this position may be important for maintaining proper folding and for stabilizing the quaternary structure. However, mutation at this site did not result in global changes in tertiary or quaternary structure as measured by Cibacron blue binding, chemical cross linking, and native gel electrophoretic analysis, leaving open the possibility of other mechanisms by which mutation of this conserved lysine residue might decrease enzyme activity.
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spelling pubmed-20965712008-02-27 Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) Basu, Saswata Murphy-Piedmonte, Deirdre M. Kirley, Terence L. Purinergic Signal Original Paper Cell membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are homooligomeric, with native quaternary structure required for maximal enzyme activity. In this study, we mutated lysine 79 in human ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3). The residue corresponding to lysine 79 in NTPDase3 is conserved in all known cell surface membrane NTPDases (NTPDase1, 2, 3, and 8), but not in the soluble, monomeric NTPDases (NTPDase5 and 6), or in the intracellular, two transmembrane NTPDases (NTPDase4 and 7). This conserved lysine is located between apyrase conserved region 1 (ACR1) and an invariant glycosylation site (N81), in a region previously hypothesized to be important for NTPDase3 oligomeric structure. This lysine residue was mutated to several different amino acids, and all mutants displayed substantially decreased nucleotidase activities. A basic amino acid at this position was found to be important for the increase of nucleotidase activity observed after treatment with the lectin, concanavalin A. After solubilization with Triton X-100, mutants showed little or no decrease in activity, unlike the wild-type enzyme, suggesting that the lysine at this position may be important for maintaining proper folding and for stabilizing the quaternary structure. However, mutation at this site did not result in global changes in tertiary or quaternary structure as measured by Cibacron blue binding, chemical cross linking, and native gel electrophoretic analysis, leaving open the possibility of other mechanisms by which mutation of this conserved lysine residue might decrease enzyme activity. Springer Netherlands 2004-12 /pmc/articles/PMC2096571/ /pubmed/18404400 http://dx.doi.org/10.1007/s11302-004-4741-8 Text en © Springer 2004
spellingShingle Original Paper
Basu, Saswata
Murphy-Piedmonte, Deirdre M.
Kirley, Terence L.
Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)
title Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)
title_full Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)
title_fullStr Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)
title_full_unstemmed Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)
title_short Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)
title_sort conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (ntpdase3)
topic Original Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2096571/
https://www.ncbi.nlm.nih.gov/pubmed/18404400
http://dx.doi.org/10.1007/s11302-004-4741-8
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