Transition-state analysis of nucleoside hydrolase from Crithidia fasciculata.
The transition state of nucleoside hydrolase from the trypanosome Crithidia fasciculata has been characterized by multiple Vmax/Km kinetic isotope effects with labeled inosine and adenosine as substrates. Nucleoside hydrolase catalyzes the hydrolysis of the N-glycosidic linkage of the commonly occurring purine and pyrimidine nucleosides, with Vmax/Km ranging over 2 orders of magnitude. The kinetic isotope effects for inosine were [1'-3H] = 1.150 +/- 0.006, [2'-3H] = 1.161 +/- 0.003, [1'-14C] = 1.044 +/- 0.004, [9-15N] = 1.026 +/- 0.004, [4'-3H] = 0.992 +/- 0.003, and [5'-3H] = 1.051 +/- 0.003. The magnitude of the kinetic isotope effects for inosine, an equivalent [1'-3H] kinetic isotope effect for the poor substrate adenosine, and the rapid equilibrium random kinetic mechanism [Parkin D, W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., & Schramm, V. L. (1991) J. Biol. Chem. (in press)] all indicate that the isotope effects are fully expressed. The kinetic and solvent deuterium isotope effects have been used to analyze the transition-state structure using bond energy bond order vibrational analysis. The transition state involves a protonated hypoxanthine leaving group with a C-N glycosidic bond elongated to approximately 2 A. The ribose group contains substantial carbocationic character, unusually strong hyperconjugation of H2', and a bond length of approximately 3 A to the incoming oxygen nucleophile. The remote isotope effect (4'-3H and 5'-3H) and the results of transition-state calculations provide the most detailed description of the steric and bonding properties of an enzyme-stabilized transition state.
Horenstein
B A
BA
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Parkin
D W
DW
Estupiñán
B
B
Schramm
V L
VL
D000241:Adenosine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D016831:Crithidia fasciculata / Q000201:enzymology*; D003903:Deuterium; D006868:Hydrolysis; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism; D007288:Inosine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism*; D009590:Nitrogen Radioisotopes; D012997:Solvents; D014316:Tritium
D002250:Carbon Radioisotopes; D007042:Hypoxanthines; D009590:Nitrogen Radioisotopes; D012997:Solvents; D014316:Tritium; D019271:Hypoxanthine; D007288:Inosine; D003903:Deuterium; D009699:N-Glycosyl Hydrolases; C096466:inosine-uridine hydrolase; D000241:Adenosine