Kinetic isotope effects of nucleoside hydrolase from Escherichia coli.
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 [1'-13C], [1-15N], [1'-2H], [2'-2H], and [5'-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, C3'-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).
Hunt
Cindy
C
Department of Chemistry, Middle Tennessee State University, Box 68, Murfreesboro, TN 37132, USA.
Gillani
Niloufar
N
Farone
Anthony
A
Rezaei
Mansoureh
M
Kline
Paul C
PC
D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D016831:Crithidia fasciculata / Q000201:enzymology / Q000235:genetics; D003903:Deuterium / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007554:Isotopes / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D009587:Nitrogen Isotopes / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D014529:Uridine / Q000737:chemistry
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D007554:Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D003903:Deuterium; D009699:N-Glycosyl Hydrolases; C422989:RihC protein, E coli; D014529:Uridine