title: Transition-state analysis of Trypanosoma cruzi uridine phosphorylase-catalyzed arsenolysis of uridine.
abstract: Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2'-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli , purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2'-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1'-(14)C = 1.103, 1,3-(15)N(2) = 1.034, 3-(15)N = 1.004, 1-(15)N = 1.030, 1'-(3)H = 1.132, 2'-(2)H = 1.086, and 5'-(3)H(2) = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S(N)2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.
authors: Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors: Rafael G
authors: RG
authors: Silva
authors: Mathew J
authors: MJ
authors: Vetticatt
authors: Emilio F
authors: EF
authors: Merino
authors: Maria B
authors: MB
authors: Cassera
authors: Vern L
authors: VL
authors: Schramm
references: Biochemistry. 1992 Jul 7;31(26):5964-73
references: 1627539
references: J Am Chem Soc. 1997 Dec 17;119(50):12079-12088
references: 19079637
references: Arch Biochem Biophys. 2005 Jan 1;433(1):2-12
references: 15581561
references: Arch Biochem Biophys. 2010 May;497(1-2):35-42
references: 20226755
references: J Am Chem Soc. 2009 Feb 18;131(6):2397-403
references: 19146405
references: J Am Chem Soc. 2007 Jun 6;129(22):7055-64
references: 17497857
references: Annu Rev Biochem. 1981;50:103-31
references: 7023356
references: Biochemistry. 2001 Jan 30;40(4):853-60
references: 11170405
references: Biochemistry. 1998 Jun 16;37(24):8615-21
references: 9628722
references: Chem Rev. 2006 Feb;106(2):506-55
references: 16464017
references: J Am Chem Soc. 2006 Nov 15;128(45):14691-6
references: 17090056
references: FEBS Lett. 1994 Nov 28;355(2):192-4
references: 7982499
references: J Am Chem Soc. 2010 Sep 8;132(35):12286-98
references: 20718419
references: Annu Rev Biochem. 1998;67:693-720
references: 9759501
references: J Mol Biol. 1999 Jan 8;285(1):149-61
references: 9878395
references: Arch Biochem Biophys. 1952 Sep;40(1):42-9
references: 12997187
references: Biochemistry. 2011 Apr 5;50(13):2701-9
references: 21348499
references: Nature. 1970 Aug 15;227(5259):680-5
references: 5432063
references: Biochemistry. 2008 Feb 26;47(8):2565-76
references: 18281957
references: Arch Biochem Biophys. 1983 Oct 15;226(2):687-92
references: 6357095
references: Biochemistry. 2011 Mar 1;50(8):1412-20
references: 21222488
references: J Am Chem Soc. 2010 Jun 30;132(25):8787-94
references: 20527751
references: Biochemistry. 2004 Feb 17;43(6):1458-68
references: 14769022
references: J Biol Chem. 1984 Feb 25;259(4):2429-34
references: 6546573
references: Biochem J. 2002 Jan 1;361(Pt 1):1-25
references: 11743878
references: J Am Chem Soc. 2010 Sep 29;132(38):13425-33
references: 20804144
references: Methods Enzymol. 1982;87:625-41
references: 7176928
references: J Mol Biol. 2010 Mar 12;396(5):1244-59
references: 20070944
references: Biochim Biophys Acta. 2002 Jul 18;1587(2-3):133-44
references: 12084455
references: Biochemistry. 2004 Jan 13;43(1):9-18
references: 14705926
references: Methods Enzymol. 1999;308:301-55
references: 10507010
references: Biochemistry. 1981 Mar 31;20(7):1790-6
references: 7013799
references: Biochemistry. 2007 May 1;46(17):5038-49
references: 17407325
references: Mol Biochem Parasitol. 1983 Jan;7(1):41-51
references: 6601772
references: Methods Enzymol. 1980;64:47-59
references: 7374457
references: Biochemistry. 2000 Nov 21;39(46):14054-64
references: 11087352
references: J Biol Chem. 1984 Apr 25;259(8):5073-7
references: 6425283
references: J Am Chem Soc. 2009 Apr 8;131(13):4685-94
references: 19292447
references: Biochemistry. 2008 Feb 26;47(8):2577-83
references: 18281958
references: Biochemistry. 1993 Dec 7;32(48):13212-9
references: 8241176
references: Biochemistry. 1975 Jun 17;14(12):2644-51
references: 1148173
references: J Biol Chem. 1984 Aug 10;259(15):9411-7
references: 6746654
references: Biochemistry. 2005 Sep 6;44(35):11647-59
references: 16128565