Guanosine-inosine-preferring nucleoside N-glycohydrolase from Crithidia fasciculata
Protozoan parasites are incapable of de novo purine biosynthesis and obtain purines by salvage pathways. A nucleoside hydrolase which prefers inosine and uridine as substrates (IU-nucleoside hydrolase) has been characterized and implicated in purine salvage in Crithidia fasciculata (Parkin, D. W., H...
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Published in | The Journal of biological chemistry Vol. 269; no. 37; pp. 23068 - 23073 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
16.09.1994
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Subjects | |
Online Access | Get full text |
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Summary: | Protozoan parasites are incapable of de novo purine biosynthesis and obtain purines by salvage pathways. A nucleoside hydrolase
which prefers inosine and uridine as substrates (IU-nucleoside hydrolase) has been characterized and implicated in purine
salvage in Crithidia fasciculata (Parkin, D. W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., and Schramm, V. L. (1991)
J. Biol. Chem. 31, 20658-20665). Treatment of C. fasciculata with inhibitors of the IU-nucleoside hydrolase did not prevent
cell growth, suggesting alternative enzymes. A guanosine-inosine-preferring enzyme (GI-nucleoside hydrolase) has been purified
from extracts of C. fasciculata and characterized. The enzyme is an oligomer of M(r) 38,500 subunits. The Vmax/Km for guanosine,
inosine, and adenosine are 3.2 x 10(6), 6.2 x 10(6), and 9.8 M-1 S-1, respectively. Deoxynucleosides, nucleotides, and pyrimidine
nucleosides are poor substrates. The pH profile for Km is independent of pH, whereas both Vmax and Vmax/Km demonstrate that
a single protonated base, pKa 7.7 is required for activity. The transition state inhibitors of IU-nucleoside hydrolase, 1,4-dideoxy-1,4-imino-1-(S)-phenyl-D-ribitol
(Horenstein, B. A., and Schramm, V. L. (1993) Biochemistry 32, 9917-9925) and p-nitrophenylriboamidrazone (Boutellier, M.,
Horenstein, B. A., Semenyaka, A., Schramm, V.L., and Ganem, B. (1994) Biochemistry 33, 3994-4000), are unexceptional inhibitors
of the GI-nucleoside hydrolase. The enzyme is inhibited by 3-deazaadenosine and 2-iodoadenosine with Km/Ki values of 145 and
61, respectively. The results demonstrate that this previously uncharacterized enzyme has distinct structure, kinetic, and
chemical mechanisms relative to IU-nucleoside hydrolase. Metabolic studies with labeled inosine as the sole purine source
indicated that the GI-enzyme is efficient for purine salvage in vivo. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(17)31620-4 |