Studies of inhibitor binding to Escherichia coli purine nucleoside phosphorylase using the transferred nuclear Overhauser effect and rotating-frame nuclear Overhauser enhancement.


:NMR studies of the adenosine analog tubercidin have been carried out in the presence of Escherichia coli purine nucleoside phosphorylase (PNP) in order to characterize the conformation of the enzyme-complexed nucleoside. Although analysis of transferred NOE data at various enzyme/inhibitor ratios indicated a predominantly syn nucleoside conformation in the enzyme-complexed state, the results, particularly the 8(1') and 8(3') NOE interactions, were not quantitatively consistent with any single bound conformation. Dissociation rate constants for the tubercidin-PNP complex were determined based on analysis of chemical shift and line width data as a function of enzyme/inhibitor ratio, Carr-Purcell-Meiboom-Gill measurements of the transverse relaxation rate as a function of pulse rate, and T1 rho experiments as a function of the spin-lock field strength. Dissociation rate constants of 2100 s-1 at 20 degrees C and 1400 s-1 at 10 degrees C were determined using the latter two methods. These rates are sufficiently high to justify the validity of the transferred NOE method for an enzyme as large as PNP. The possible significance of spin diffusion was investigated by the use of the deuterated analog [2'-2H]tubercidin, for which many of the intraligand spin diffusion pathways are eliminated, and by performing a series of transferred ROE experiments. A comparison of data obtained using transferred NOE and ROE measurements provides a basis for separating direct and indirect relaxation pathways. Both approaches indicated that the relatively significant 8(3') NOE interaction was not dominated by spin diffusion. Furthermore, analysis of chemical shift and transverse relaxation data for the tubercidin H-2 resonance gave inconsistent results for the chemical shift of the bound species and was inconsistent with the assumption of a single, bound conformation. These results were interpreted in terms of a 2:1 ratio of a syn, 3'-exo:anti, 3'-endo geometry for bound tubercidin. Ligand competition experiments using 9-deazainosine show that all of the tubercidin TRNOE effects are reversed by addition of the second nucleoside, suggesting that the TRNOE data for tubercidin arise due to interactions at the active sites of PNP rather than as a consequence of nonspecific binding to the enzyme.






Perlman ME,Davis DG,Koszalka GW,Tuttle JV,London RE




Has Abstract


1994-06-21 00:00:00












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    pub_type: 杂志文章


    authors: Retzios AD,Markland FS Jr

    更新日期:1992-05-19 00:00:00

  • Spin labeling analysis of structure and dynamics of the Na(+)/proline transporter of Escherichia coli.

    abstract::With respect to the functional importance attributed to the N-terminal part of the Na(+)/proline transporter of Escherichia coli (PutP), we report here on the structural arrangement and functional dynamics of transmembrane domains (TMs) II and III and the adjoining loop regions. Information on membrane topography was ...


    pub_type: 杂志文章


    authors: Wegener C,Tebbe S,Steinhoff HJ,Jung H

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  • Crystal structure of arginase from Plasmodium falciparum and implications for L-arginine depletion in malarial infection .

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    pub_type: 杂志文章


    authors: Dowling DP,Ilies M,Olszewski KL,Portugal S,Mota MM,Llinás M,Christianson DW

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    pub_type: 杂志文章


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    pub_type: 杂志文章


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    pub_type: 杂志文章


    authors: Han J,Burke JM

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    pub_type: 杂志文章


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    更新日期:1995-03-14 00:00:00

  • Discovery of a novel L-lyxonate degradation pathway in Pseudomonas aeruginosa PAO1.

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  • Crystal structure of guanosine-free ribonuclease T1, complexed with vanadate (V), suggests conformational change upon substrate binding.

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    pub_type: 杂志文章


    authors: Kostrewa D,Choe HW,Heinemann U,Saenger W

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  • Structural Diversity of Amyloid Fibrils and Advances in Their Structure Determination.

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    pub_type: 杂志文章


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    pub_type: 杂志文章


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