Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10537312 | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics | 2005 | 10 Pages |
Abstract
rihC is one of a group of three ribonucleoside hydrolases found in Escherichia coli (E. coli). The enzyme catalyzes the hydrolysis of selected nucleosides to ribose and the corresponding base. A family of Vmax/Km kinetic isotope effects using uridine labeled with stable isotopes, such as 2H, 13C, and 15N, were determined by liquid chromatography/mass spectrometry (LC/MS). The kinetic isotope effects were 1.012 ± 0.006, 1.027 ± 0.005, 1.134 ± 0.007, 1.122 ± 0.008, and 1.002 ± 0.004 for [1â²-13C], [1-15N], [1â²-2H], [2â²-2H], and [5â²-2H2] uridine, respectively. A transition state based upon a bond-energy bond-order vibrational analysis (BEBOVIB) of the observed kinetic isotope effects is proposed. The main features of this transition state are activation of the heterocyclic base by protonation of/or hydrogen bonding to O2, an extensively broken C-N glycosidic bond, formation of an oxocarbenium ion in the ribose ring, C3â²-exo ribose ring conformation, and almost no bond formation to the attacking nucleophile. The proposed transition state for the prokaryotic E. coli nucleoside hydrolase is compared to that of a similar enzyme isolated from Crithidia fasciculata (C. fasciculata).
Keywords
OctadecylODSIPTGTRISGC/MSLC/MSKinetic isotope effectkinetic isotope effectsSDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresisisopropyl β-D-thiogalactopyranosideTris(hydroxymethyl)aminomethaneTransition stateSIMLuria–Bertaniselective ion monitoringnucleoside hydrolaseLiquid chromatography/mass spectrometrygas chromatography/mass spectrometryKIE
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Cindy Hunt, Niloufar Gillani, Anthony Farone, Mansoureh Rezaei, Paul C. Kline,