Article

Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6-phosphogluconate dehydrogenase.

Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Italy.
FEBS Journal (Impact Factor: 3.99). 01/2008; 274(24):6426-35. DOI: 10.1111/j.1742-4658.2007.06160.x
Source: PubMed

ABSTRACT 6-Phosphogluconate dehydrogenase is a potential target for new drugs against African trypanosomiasis. Phosphorylated aldonic acids are strong inhibitors of 6-phosphogluconate dehydrogenase, and 4-phospho-d-erythronate (4PE) and 4-phospho-d-erythronohydroxamate are two of the strongest inhibitors of the Trypanosoma brucei enzyme. Binding of the substrate 6-phospho-d-gluconate (6PG), the inhibitors 5-phospho-d-ribonate (5PR) and 4PE, and the coenzymes NADP, NADPH and NADP analogue 3-amino-pyridine adenine dinucleotide phosphate to 6-phospho-d-gluconate dehydrogenase from T. brucei was studied using isothermal titration calorimetry. Binding of the substrate (K(d) = 5 microm) and its analogues (K(d) =1.3 microm and K(d) = 2.8 microm for 5PR and 4PE, respectively) is entropy driven, whereas binding of the coenzymes is enthalpy driven. Oxidized coenzyme and its analogue, but not reduced coenzyme, display a half-site reactivity in the ternary complex with the substrate or inhibitors. Binding of 6PG and 5PR poorly affects the dissociation constant of the coenzymes, whereas binding of 4PE decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar manner, the K(d) value of 4PE decreases by two orders of magnitude in the presence of the coenzymes. The results suggest that 5PR acts as a substrate analogue, whereas 4PE mimics the transition state of dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the mechanism of the enzyme, suggesting that the inhibitor forces the catalytic lysine 185 into the protonated state.

Download full-text

Full-text

Available from: Stefania Hanau, Aug 28, 2014
0 Followers
 · 
100 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Elucidation of the energetic principles of binding affinity and specificity is a central task in many branches of current sciences: biology, medicine, pharmacology, chemistry, material sciences, etc. In biomedical research, integral approaches combining structural information with in-solution biophysical data have proved to be a powerful way toward understanding the physical basis of vital cellular phenomena. Isothermal titration calorimetry (ITC) is a valuable experimental tool facilitating quantification of the thermodynamic parameters that characterize recognition processes involving biomacromolecules. The method provides access to all relevant thermodynamic information by performing a few experiments. In particular, ITC experiments allow to by-pass tedious and (rarely precise) procedures aimed at determining the changes in enthalpy and entropy upon binding by van't Hoff analysis. Notwithstanding limitations, ITC has now the reputation of being the ''gold standard'' and ITC data are widely used to validate theoretical predictions of thermodynamic parameters, as well as to benchmark the results of novel binding assays. In this paper, we discuss several publications from 2007 reporting ITC results. The focus is on applications in biologically oriented fields. We do not intend a comprehensive coverage of all newly accumulated information. Rather, we emphasize work which has captured our attention with originality and far-reaching analysis, or else has provided ideas for expanding the potential of the method.
    Journal of Molecular Recognition 09/2008; 21(5):289-312. DOI:10.1002/jmr.909 · 2.34 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the oxidative decarboxylation of 6-phosphogluconate, making ribulose 5-phosphate, along with the reduction of NADP(+) to NADPH and the release of CO(2). Here, we report the first apo-form crystal structure of the pathogenic Klebsiella pneumoniae 6PGDH (Kp6PGDH) and the structures of the highly homologous Escherichia coli K12 6PGDH (Ec6PGDH) complexed with substrate, substrate/NADPH and glucose at high resolution. The binding of NADPH to one subunit of the homodimeric structure triggered a 10 degrees rotation and resulting in a 7A movement of the coenzyme-binding domain. The coenzyme was thus trapped in a closed enzyme conformation, in contrast to the open conformation of the neighboring subunit. Comparison of our Ec/Kp6PGDH structures with those of other species illustrated how the domain conformation can be affected upon binding of the coenzyme, which in turn gives rise to concomitant movements of two important NADP(+)-interacting amino acids, M14 and N102. We propose that the catalysis follows an ordered binding mechanism with alternating conformational changes in the corresponding subunits, involving several related amino acid residues.
    Journal of Structural Biology 09/2009; 169(1):25-35. DOI:10.1016/j.jsb.2009.08.006 · 3.37 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG (D)(V/K) and (D)(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases (D)(V/K) to 2.84 and decreases (D)(V) to 1.38. A similar increase of (T)(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the K(m) of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH.
    Journal of Biological Chemistry 05/2010; 285(28):21366-71. DOI:10.1074/jbc.M110.105601 · 4.60 Impact Factor