GUANOSINE-INOSINE-PREFERRING NUCLEOSIDE N-GLYCOHYDROLASE FROM CRITHIDIA-FASCICULATA

Protozoan parasites are incapable of de novo purine biosynthesis and o btain purines by salvage pathways. A nucleoside hydrolase which prefer s inosine and uridine as substrates (IU-nucleoside hydrolase) has been characterized and implicated in purine salvage in Crithidia fascicula ta (Parkin, D. W., Horenstein, B. A., Abdulah, D. R., Estupinan, B., a nd 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-inos ine-preferring enzyme (GI-nucleoside hydrolase) has been purified from extracts of C. fasciculata and characterized. The enzyme is an oligom er of M(r) 38,500 subunits. The V-max/K-m for guanosine, inosine, and adenosine are 3.2 x 10(6), 6.2 x 10(6), and 9.8 m(-1) s(-1), respectiv ely. Deoxynucleosides, nucleotides, and pyrimidine nucleosides are poo r substrates. The pH profile for K-m is independent of pH, whereas bot h V-max and V-max/K-m demonstrate that a single protonated base, pK(al pha) 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, 9917-992 5) and p-nitrophenylriboamidrazone (Boutellier, M., Horenstein, B. A., Semenyaka, A., Schramm, V.L., and Ganem, B. (1994) Biochemistry 33, 3 994-4000), 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 t hat this previously uncharacterized enzyme has distinct structure, kin etic, and chemical mechanisms relative to IU-nucleoside hydrolase. Met abolic studies with labeled inosine as the sole purine source indicate d that the GI-enzyme is efficient for purine salvage in vivo.