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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

An NMR study of biological phosphate interactions

Wilson, Henry R. January 1986 (has links)
An investigation is made of the interactions between a variety of small molecule phosphates and amines, and in particular of triethylenetetramine with hexametaphosphate, pyrophosphate, tripolyphosphate, ADP and ATP and of cyclic triethylenetriamine with hexametaphosphate. For each phosphate/amine pair, observed changes in the pH dependence of <sup>1</sup>H and <sup>31</sup>P nmr chemical shift are used to calculate the effect of interaction on the pK<sub>a</sub> of the two species present and an association constant is evaluated at one particular pH value. These data are then combined to evaluate association constants for a variety of protonation states of the phosphate and amine. Strength of association is interpreted in terms of electrostatic charge and its spatial distribution, comparison is made with Mg<sup>2+</sup> and Ca<sup>2+</sup> complexes and biological relevance is discussed. The interaction of yeast PGK with its substrates is investigated by using the (500MHz and 300MHz) <sup>1</sup>H nmr spectrum of the enzyme and (250MHz) <sup>31</sup>P nmr spectra of its substrates. On the basis of pH dependence, comparison with the spectrum of horse PGK and T<sub>2</sub> relaxation data,<sup>1</sup>H nmr resonances are assigned to surface residues. In particular, resonances are assigned to the three active site histidines (nos. 62,167 and 170), and to the exposed histidine and methionine residues. A series of comparatively well-resolved upfield shifted aromatic peaks, including a relatively exposed tyrosine peak is tentatively assigned to the interdomain hinge region of the protein. The effects of MgADP, MgATP and the non-reacting nucleotide analogue MgAMPPNP on the yeast PGK spectrum are characterised. The line-broadening reagent Gd<sup>3+</sup>is used to distinguish between direct influence and conformational effects. The triose phosphate substrate 3-PG is added to the enzyme as are the anionic probes [Co(CN)<sub>6</sub>]<sup3-</sup> , [Cr(CN)<sub>6</sub>]<sup3-</sup>and [Fe(CN)<sub>6</sub>]<sup3-</sup> I and the nature of anion binding in the active groove of the protein and elsewhere on its surface is investigated. Movement of the two PGK domains is investigated in three models of the ternary complex: (i) with MgAMPPNP and 3-PG, (ii) with the elongated nucleotide A4P, and (iii) with a mutant form of the enzyme (his388 -gln388). The PGK/substrate interaction is interpreted in terms of the phosphate/amine model.

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