title: Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases.
abstract: Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to 2'-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C(439)) in α2 by a stable diferric tyrosyl radical (Y(122)•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y(122)•→ W(48)→ Y(356) in β2 to Y(731)→ Y(730)→ C(439) in α2. To study the details of this process, 3-aminotyrosine (NH(2)Y) has been site-specifically incorporated in place of Y(356) of β. The resulting protein, Y(356)NH(2)Y-β2, and the previously generated proteins Y(731)NH(2)Y-α2 and Y(730)NH(2)Y-α2 (NH(2)Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2-0.7 s(-1). Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH(2)Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH(2)Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH(2)Y• formation is biphasic (k(fast) of 9-46 s(-1) and k(slow) of 1.5-5.0 s(-1)) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH(2)Y• formation, while the k(cat) of ~0.5 s(-1) is proposed to be associated with rate-limiting oxidation by NH(2)Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH(2)Y-α2 and Y(731)NH(2)Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.
authors: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
authors: Ellen C
authors: EC
authors: Minnihan
authors: Mohammad R
authors: MR
authors: Seyedsayamdost
authors: Ulla
authors: U
authors: Uhlin
authors: JoAnne
authors: J
authors: Stubbe
references: Biochemistry. 2011 Mar 1;50(8):1403-11
references: 21182280
references: J Mol Biol. 2010 Jan 15;395(2):361-74
references: 19852970
references: J Biol Chem. 1966 Jun 10;241(11):2540-9
references: 5330120
references: J Am Chem Soc. 2010 Nov 3;132(43):15368-79
references: 20929229
references: J Phys Chem B. 2008 Sep 18;112(37):11827-37
references: 18717535
references: Biochemistry. 2009 Dec 29;48(51):12125-32
references: 19916558
references: J Biol Chem. 1989 Jul 25;264(21):12249-52
references: 2663852
references: J Bacteriol. 1986 Feb;165(2):363-6
references: 3511029
references: J Am Chem Soc. 2006 Mar 1;128(8):2522-3
references: 16492021
references: J Am Chem Soc. 2007 Dec 5;129(48):15060-71
references: 17990884
references: Biochemistry. 1993 Nov 30;32(47):12749-60
references: 8251496
references: Biochemistry. 1987 Jun 16;26(12):3408-16
references: 3307907
references: Biochemistry. 2003 Dec 16;42(49):14541-52
references: 14661967
references: J Am Chem Soc. 2003 Sep 3;125(35):10506-7
references: 12940718
references: Methods Enzymol. 2009;462:45-76
references: 19632469
references: Biochemistry. 1999 Jul 20;38(29):9495-507
references: 10413527
references: J Biol Chem. 1973 Jul 10;248(13):4591-601
references: 4578086
references: Biochemistry. 1999 Jan 26;38(4):1221-33
references: 9930982
references: Methods Enzymol. 1995;258:278-303
references: 8524156
references: Methods. 2005 Jul;36(3):227-38
references: 16076448
references: Chem Rev. 1998 Apr 2;98(2):705-762
references: 11848913
references: Nature. 1994 Aug 18;370(6490):533-9
references: 8052308
references: J Am Chem Soc. 2007 Feb 28;129(8):2226-7
references: 17279757
references: Nature. 1990 Jun 14;345(6276):593-8
references: 2190093
references: J Am Chem Soc. 2006 Feb 8;128(5):1562-8
references: 16448127
references: Chem Rev. 2003 Jun;103(6):2167-201
references: 12797828
references: Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9
references: 2025413
references: J Am Chem Soc. 2009 Nov 4;131(43):15729-38
references: 19821570
references: Annu Rev Biochem. 1998;67:71-98
references: 9759483
references: J Am Chem Soc. 2010 Jun 23;132(24):8385-97
references: 20518462
references: Biochemistry. 2003 Sep 2;42(34):10071-83
references: 12939135
references: Biochemistry. 2009 Dec 15;48(49):11622-9
references: 19899770
references: Annu Rev Biochem. 2006;75:681-706
references: 16756507
references: Biochemistry. 1997 Dec 2;36(48):14985-91
references: 9398223
references: J Bacteriol. 1985 Jul;163(1):238-42
references: 2989245