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., 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.
Estupiñán
B
B
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Schramm
V L
VL
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D016831:Crithidia fasciculata / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006151:Guanosine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007288:Inosine / Q000378:metabolism*; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012255:Ribitol / Q000031:analogs & derivatives / Q000494:pharmacology; D013379:Substrate Specificity
C089322:phenyliminoribitol; D006151:Guanosine; D012255:Ribitol; D007288:Inosine; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C041035:inosine-guanosine hydrolase