Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism.
Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
D W
DW
Parkin
B A
BA
Horenstein
D R
DR
Abdulah
B
B
Estupiñán
V L
VL
Schramm
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D016831:Crithidia fasciculata / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011687:Purines / Q000378:metabolism; D012997:Solvents; D013379:Substrate Specificity
D011687:Purines; D012997:Solvents; D009699:N-Glycosyl Hydrolases; C096466:inosine-uridine hydrolase