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., Estupinan, 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 V(max)/K(m) for guanosine, inosine, and adenosine are 3.2 x 106, 6.2 x 106, and 9.8 M-1 s-1, respectively. Deoxynucleosides, nucleotides, and pyrimidine nucleosides are poor substrates. The pH profile for K(m) is independent of pH, whereas both V(max) and V(max)/K(m) demonstrate that a single protonated base, pK(α) 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 K(m)/K(i) 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.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Sep 16 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology