Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei: Purification, specificity, and kinetic mechanism
Trypanosomes have no de novo purine biosynthesis and thus depend upon salvage pathways to obtain purines for their metabolic pathways and for the biosynthesis of nucleic acids. An inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-nucleoside hydrolase) from the African trypanosome Tryp...
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| Formato: | Journal Article |
| Lenguaje: | Inglés |
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Elsevier
1996
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| Acceso en línea: | https://hdl.handle.net/10568/32992 |
| _version_ | 1855540699336802304 |
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| author | Parkin, D.W. |
| author_browse | Parkin, D.W. |
| author_facet | Parkin, D.W. |
| author_sort | Parkin, D.W. |
| collection | Repository of Agricultural Research Outputs (CGSpace) |
| description | Trypanosomes have no de novo purine biosynthesis and thus depend upon salvage pathways to obtain purines for their metabolic pathways and for the biosynthesis of nucleic acids. An inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-nucleoside hydrolase) from the African trypanosome Trypanosoma brucei brucei represents 0.2 percent of the soluble protein in this organism. The enzyme has been purified over 400-fold to>95 percent homogeneity from the bloodstream form of this parasite. IAG-nucleoside hydrolase is a dimer of Mr 36,000 subunits. The k cat/km for inosine, adenosine, and guanosine are 1.9 x 10(6), 1.2 x10(6), and 0.83 x 10(6) M-1 d-1, respectively. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The turnover rate for inosine at 30 degree C is 34 s-1. Pyrimidine nucleosides are poor substrates with k cat/Km values of approximately 10(3) M-1 s-1. Deoxynucleosides are also poor substrates with kcat/Km values near 10(2) M-1 s-1. AMP is not a detectable substrate and there is no measurable purine nucleoside phosphorylase activity. 3-Deazaadenosine, 7-deazaadenosine (tubercidin), and formycin B are competitive inhibitors with K is of 1.8, 59, and 13 M, respectively. The Km shows a slight dependence on pH with a pH optimum around 7. The V max/km data indicate there are two ionizable enzymatic groups, pKa 8.6, required for the formation of the Michaelis complex. The V max data indicate three ionizable groups in-volved in catalysis. Two essential groups exhibit pKa values of 8.8, and a third group with a pKa of 6.5 increases the V max an additional 10-fold. All three groups must be protonated for optimum catalytic activity. |
| format | Journal Article |
| id | CGSpace32992 |
| institution | CGIAR Consortium |
| language | Inglés |
| publishDate | 1996 |
| publishDateRange | 1996 |
| publishDateSort | 1996 |
| publisher | Elsevier |
| publisherStr | Elsevier |
| record_format | dspace |
| spelling | CGSpace329922024-11-18T08:44:26Z Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei: Purification, specificity, and kinetic mechanism Parkin, D.W. trypanosoma brucei nucleosides hydrolases Trypanosomes have no de novo purine biosynthesis and thus depend upon salvage pathways to obtain purines for their metabolic pathways and for the biosynthesis of nucleic acids. An inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-nucleoside hydrolase) from the African trypanosome Trypanosoma brucei brucei represents 0.2 percent of the soluble protein in this organism. The enzyme has been purified over 400-fold to>95 percent homogeneity from the bloodstream form of this parasite. IAG-nucleoside hydrolase is a dimer of Mr 36,000 subunits. The k cat/km for inosine, adenosine, and guanosine are 1.9 x 10(6), 1.2 x10(6), and 0.83 x 10(6) M-1 d-1, respectively. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The turnover rate for inosine at 30 degree C is 34 s-1. Pyrimidine nucleosides are poor substrates with k cat/Km values of approximately 10(3) M-1 s-1. Deoxynucleosides are also poor substrates with kcat/Km values near 10(2) M-1 s-1. AMP is not a detectable substrate and there is no measurable purine nucleoside phosphorylase activity. 3-Deazaadenosine, 7-deazaadenosine (tubercidin), and formycin B are competitive inhibitors with K is of 1.8, 59, and 13 M, respectively. The Km shows a slight dependence on pH with a pH optimum around 7. The V max/km data indicate there are two ionizable enzymatic groups, pKa 8.6, required for the formation of the Michaelis complex. The V max data indicate three ionizable groups in-volved in catalysis. Two essential groups exhibit pKa values of 8.8, and a third group with a pKa of 6.5 increases the V max an additional 10-fold. All three groups must be protonated for optimum catalytic activity. 1996-09 2013-07-03T05:25:53Z 2013-07-03T05:25:53Z Journal Article https://hdl.handle.net/10568/32992 en Open Access Elsevier Parkin, D. W. (1996). Purine-specific Nucleoside N-Ribohydrolase from Trypanosoma brucei brucei. Journal of Biological Chemistry 271(36): 21713–21719. Elsevier BV. https://doi.org/10.1074/jbc.271.36.21713 |
| spellingShingle | trypanosoma brucei nucleosides hydrolases Parkin, D.W. Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei: Purification, specificity, and kinetic mechanism |
| title | Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei: Purification, specificity, and kinetic mechanism |
| title_full | Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei: Purification, specificity, and kinetic mechanism |
| title_fullStr | Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei: Purification, specificity, and kinetic mechanism |
| title_full_unstemmed | Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei: Purification, specificity, and kinetic mechanism |
| title_short | Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei: Purification, specificity, and kinetic mechanism |
| title_sort | purine specific nucleoside n ribohydrolase from trypanosoma brucei brucei purification specificity and kinetic mechanism |
| topic | trypanosoma brucei nucleosides hydrolases |
| url | https://hdl.handle.net/10568/32992 |
| work_keys_str_mv | AT parkindw purinespecificnucleosidenribohydrolasefromtrypanosomabruceibruceipurificationspecificityandkineticmechanism |