Kinetic isotope effects of nucleoside hydrolase from Escherichia coli

✍ Scribed by Cindy Hunt; Niloufar Gillani; Anthony Farone; Mansoureh Rezaei; Paul C. Kline

• Publisher: Elsevier Science
• Year: 2005
• Tongue: English
• Weight: 405 KB
• Volume: 1751
• Category: Article
• ISSN: 1570-9639
• DOI: 10.1016/j.bbapap.2005.06.001

✦ Synopsis

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 V max /K m kinetic isotope effects using uridine labeled with stable isotopes, such as 2 H, 13 C, and 15 N, were determined by liquid chromatography/mass spectrometry (LC/MS). The kinetic isotope effects were 1.012 T 0.006, 1.027 T 0.005, 1.134 T 0.007, 1.122 T 0.008, and 1.002

and [5V-2 H 2 ] 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, C3V-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).