PubMed · 15529160
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Irving W Wainer. 2004. Finding time for allosteric interactions.. https://doi.org/10.1038/nbt1104-1376
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PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO4(2-) ions alone and with ATP, Cd2+ and SO4(2-) ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the beta- and gamma-phosphates of ATP. A SO4(2-) ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO4(2-) binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO4(2-) stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO4(2-) and affect the enzymatic activity. The results in the present paper suggest that this new SO4(2-)-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.
Patented signal analytic algorithms applied to hydrophobically transformed, numerical amino acid sequences have previously been used to design short, protein-targeted, L or D retro-inverso peptides. These peptides have demonstrated allosteric and/or indirect agonist effects on a variety of G-protein and tyrosine kinase coupled membrane receptors with 30% to over 80% hit rates. Here we extend these approaches to a globular protein target. We designed eight peptide ligands targeting an ELISA antibody responsive protein, beta-galactosidase, betaGAL. Three of the eight 14mer peptides allosterically activated betaGAL with ELISA methodology. Using Bayesian statistics, this 38% hit rate would have occurred 2 x 10(-9) by chance. These peptides demonstrated binding site competitive or noncompetitive interactions, suggesting allosteric site multiplicity with respect to their betaGAL binding-mediated ELISA signal. Kinetic studies demonstrated the temperature dependence of the betaGAL peptide binding functions. Using the van't Hoff relation, we found evidence for enthalpy-entropy compensation. This relation is often found for hydrophobic interactions in aqueous media, and is consistent with the postulated hydrophobic series encoding underlying our protein-targeted, peptide design methods. It appears that our algorithmic, hydrophobic autocovariance eigenvector template approach to the design of allosteric peptides targeting membrane receptors may also be applicable to the design of peptide ligands targeting nonmembrane involved globular proteins.
Cyclodextrins are useful models in the study of hydrogen bonded water clusters. In alpha-cyclodextrin hexahydrate (alpha-CD.6H2O), water molecules are ordered and occupy well-defined positions whereas in the larger beta-cyclodextrin dodecahydrate (beta-CD.12H2O), there is considerable disorder with water molecules freely arranged over several possible sites. Here it is shown that beta-CD exhibits substantial structural flexibility and proton mobility compared with alpha-CD which is relatively very rigid and exhibits negligible short-range protonic conduction. These properties are directly controlled by the effective dielectric constant of the molecule, which is determined by the rotational freedom of water molecules in the hydrogen bond network. This model may be relevant to proteins where water clusters of this kind are found on the protein surface and occasionally in the protein interior. The case of thrombin, an allosteric enzyme incorporating a network of 20 internal hydrogen bonded water molecules, is discussed.