Nucleophilic participation in the transition state for human thymidine phosphorylase

被引:60
作者
Birck, MR [1 ]
Schramm, VL [1 ]
机构
[1] Yeshiva Univ Albert Einstein Coll Med, Dept Biochem, Bronx, NY 10461 USA
关键词
D O I
10.1021/ja039260h
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Recombinant human thymidine phosphorylase catalyzes the reaction of arsenate with thymidine to form thymine and 2-deoxyribose 1-arsenate, which rapidly decomposes to 2-deoxyribose and inorganic arsenate. The transition-state structure of this reaction was determined using kinetic isotope effect analysis followed by computer modeling. Experimental kinetic isotope effects were determined at physiological pH and 37 degreesC. The extent of forward commitment to catalysis was determined by pulse-chase experiments to be 0.70%. The intrinsic kinetic isotope effects for [1'-H-3]-, [2'R-H-3]-, [2'S-H-3]-, [4'-H-3]-, [5'-3H]-,[1'-C-14]-, and [1-N-15]-thymidines were determined to be 0.989 +/- 0.002, 0.974 +/- 0.002, 1.036 +/- 0.002, 1.020 +/- 0.003, 1.061 +/- 0.003, 1.139 +/- 0.005, and 1.022 +/- 0.005, respectively. A computer-gene rated model, based on density functional electronic structure calculations, was fit to the experimental isotope effect. The structure of the transition state confirms that human thymidine phosphorylase proceeds through an S(N)2-like transition state with bond orders of 0.50 to the thymine leaving group and 0.33 to the attacking oxygen nucleophile. The reaction differs from the dissociative transition states previously reported for N-ribosyl transferases and is the first demonstration of a nucleophilic transition state for an N-ribosyl transferase. The large primary C-14 isotope effect of 1.139 can occur only in nucleophilic displacements and is the largest 14C primary isotope effect reported for an enzymatic reaction. A transition state structure with substantial bond order to the attacking nucleophile and leaving group is confirmed by the slightly inverse 1'-H-3 isotope effect, demonstrating that the transition state is compressed by the impinging steric bulk of the nucleophile and leaving group.
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页码:2447 / 2453
页数:7
相关论文
共 69 条
[1]   ISOEFF98. A program for studies of isotope effects using Hessian modifications [J].
Anisimov, V ;
Paneth, P .
JOURNAL OF MATHEMATICAL CHEMISTRY, 1999, 26 (1-3) :75-86
[2]  
[Anonymous], 1998, Patent, Patent No. [5744475, US5744475]
[3]   PURIFICATION AND CHARACTERIZATION OF URIDINE AND THYMIDINE PHOSPHORYLASE FROM LACTOBACILLUS-CASEI [J].
AVRAHAM, Y ;
GROSSOWICZ, N ;
YASHPHE, J .
BIOCHIMICA ET BIOPHYSICA ACTA, 1990, 1040 (02) :287-293
[4]  
BAJORATH J, 1993, PROTEIN SCI, P2
[5]   Purine nucleoside phosphorylase from Mycobacterium tuberculosis.: Analysis of inhibition by a transition-state analogue and dissection by parts [J].
Basso, LA ;
Santos, DS ;
Shi, WX ;
Furneaux, RH ;
Tyler, PC ;
Schramm, VL ;
Blanchard, JS .
BIOCHEMISTRY, 2001, 40 (28) :8196-8203
[6]  
BENDER ML, 1957, J AM CHEM SOC, V76, P5649
[7]   Transition state structure for the hydrolysis of NAD+ catalyzed by diphtheria toxin [J].
Berti, PJ ;
Blanke, SR ;
Schramm, VL .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1997, 119 (50) :12079-12088
[8]   Ricin A-chain: Kinetic isotope effects and transition state structure with stem-loop RNA [J].
Chen, XY ;
Berti, PJ ;
Schramm, VL .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2000, 122 (08) :1609-1617
[9]   Transition-state analysis for depurination of DNA by ricin A-chain [J].
Chen, XY ;
Berti, PJ ;
Schramm, VL .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2000, 122 (28) :6527-6534
[10]   THE RELATION BETWEEN THE DIVERGENCE OF SEQUENCE AND STRUCTURE IN PROTEINS [J].
CHOTHIA, C ;
LESK, AM .
EMBO JOURNAL, 1986, 5 (04) :823-826