The microdetermination of 2,3-diphosphoglycerate

A procedure for microestimation of 2,3-diphosphoglycerate, utilizing its role as coenzyme in the phosphoglycerate mutase reaction is described. The coenzymic activity was determined by assaying phosphoglycerate mutase polarimetrically without a coupled enzyme. This method is applicable to samples containing as little as 0.002 μmole of 2,3-diphosphoglycerate/ml. The content in various biological extracts was determined.     

Enzymatic degradation of cyclic 2,3-diphosphoglycerate to 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum.

2,3-Diphosphoglycerate (2,3-DPG) has been found to be the product of the enzymatic degradation of cyclic 2,3-diphosphoglycerate (cDPG) in the archaebacterium Methanobacterium thermoautotrophicum delta H. Although 2,3-DPG has not previously been detected as a major soluble component of M. thermoautotrophicum, large pools accumulated at an incubation temperature of 50 degrees C (below the optimum growth temperature of 62 degrees C). Under these conditions, cellular activity was significantly decreased; a return of the culture to the optimum growth temperature restored the 2,3-DPG pool back to original low levels and caused steady-state cDPG levels to increase again. While 13CO2-pulse/12CO2-chase experiments at 50 degrees C showed that the cDPG turned over, the appearance of 2,3-DPG at NMR-visible concentrations required at least 10 h. Production of 2,3-DPG in vivo was prevented by exposure of the cells to O2. The enzyme responsible for this hydrolysis of cDPG was purified by affinity chromatography and appears to be a 33-kDa protein. Activity was detected in the presence of oxygen and was enhanced by a solution of 1 M KCl, 25 mM MgCl2, and dithiothreitol. Both Km and Vmax have been determined at 37 degrees C; kinetics also indicate that in vitro the product, 2,3-DPG, is an inhibitor of cDPG hydrolysis. These findings are discussed in view of a proposed role for cDPG in methanogens.     

The binding of 2,3-diphosphoglycerate as a conformational probe of human hemoglobins

Since 2,3-diphosphoglyeerate preferentially binds to deoxygenated hemoglobin A, this binding reaction can be used to detect the change in quaternary conformation of hemoglobin associated with the change in ligand state of the hemes. We have studied the binding to two M hemoglobins (M_HydePark, M_Milwaukee-1) that have the substituted chains in the ferric state, as well as to the mixed liganded hybrids α1₂β² and α²β1₂ (1 heme in cyanmet form) prepared from hemoglobins A and H. The studies demonstrate that when these hemoglobin variants and derivatives are deoxygenated, they bind the organic phosphate to an extent similar, but not identical, to that for fully deoxygenated hemoglobin A. The results indicate that removal of ligand from only two of the four hemes results in a change in quaternary structure to a deoxy-like conformation.     

Comparative studies of unfolding and binding of ligands to human serum albumin in the presence of fatty acid: spectroscopic approach

This study was undertaken to investigate the influence of fatty acid binding on the unfolding of HSA and how the fatty acid molecules can influence and/or compete with other ligand molecules bound to the protein. The equilibrium unfolding of fatted and fatty acid free HSA was measured by overlapping of unfolding transition curves monitored by different probes for secondary and tertiary structure and determining changes in free energy of unfolding. Proteins stability was studied by fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism techniques. We have suggested a "molten globule" like intermediate state of HSA at a fairly high concentration of GnHCl (3.2 for fatty acid free and 3.6 for fatted). The free energy of stabilization (DeltaG(D)(H2O)) in the presence of fatty acid was found to be 900 cal mol(-1). We also analyze the effects of fatty acid on binding of ligands using spectroscopic technique and reported the equilibrium constants and free energies obtained from the binding and unfolding experiments.     

pH dependence of 2,3-diphosphoglycerate binding to human hemoglobin A0 at 21.5 degrees C

Rate equilibrium dialysis was used to measure the binding of 2,3-diphosphoglycerate (DPG) to human oxy- and deoxyhemoglobin A0 over the range pH 5-9, at 21.5 degrees C. This approach yielded an accurate, precise, and self-consistent set of model-independent association constants. These data were successfully fitted to a thermodynamic model which is functionally similar to a Hill equation. The isotherms generated by this fitting procedure appear to intersect at low pH and converge at high pH. This apparent convergence at high pH is consistent with results obtained by oxygen equilibria studies performed under conditions of saturating DPG. These calculated isotherms were used to determine the enhancement of the Bohr effect as a function of pH. These results are consistent with data obtained by pH stat measurements by other investigators. This paper presents the first in a series of studies that will provide a systematic characterization of the interaction between hemoglobin and DPG.     

Response of erythrocytic 2,3-diphosphoglycerate to strenuous exercise.

Since increases of erythrocytic 2,3-diphosphoglycerate (2,3-DPG) have been shown to enhance the release of oxygen from hemoglobin, experiments were designed to evaluate the response of 2,3-DPG to two different work-loads in 13 fasted human subjects. No signficant mean change in 2.3-DPG was found following 16 min of strenuous exercise on a bicycle ergometer, but when the subjects were subjected later to a greater workload for 20 min, there was a significant mean decrease in 2.3-DPG despite much individual variation. In addition, there was a significant positive correlation of 2.3-DPG reduction with increases in postexercise lactate, and a significant inverse correlation of oxygen consumption during exercise with postexercise lactate. The data suggest that the 2.3-DPG mechanism may not be compensating in exercise when the workload requires a preponderance of anaerobic metabolism promoting lactacidaemia.     

Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease

The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.     

Computational analysis of binding of P1 variants to trypsin

The binding of P1 variants of bovine pancreatic trypsin inhibitor (BPTI) to trypsin has been investigated by means of molecular dynamics simulations. The specific interaction formed between the amino acid at the primary binding (P1) position of the binding loop of BPTI and the specificity pocket of trypsin was estimated by use of the linear interaction energy (LIE) method. Calculations for 13 of the naturally occurring amino acids at the P1 position were carried out, and the results obtained were found to correlate well with the experimental binding free energies. The LIE calculations rank the majority of the 13 variants correctly according to the experimental association energies and the mean error between calculated and experimental binding free energies is only 0.38 kcal/mole, excluding the Glu and Asp variants, which are associated with some uncertainties regarding protonation and the possible presence of counter-ions. The three-dimensional structures of the complex with three of the P1 variants (Asn, Tyr, and Ser) included in this study have not at present been solved by any experimental techniques and, therefore, were modeled on the basis of experimental data from P1 variants of similar size. Average structures were calculated from the MD simulations, from which specific interactions explaining the broad variation in association energies were identified. The present study also shows that explicit treatment of the complex water-mediated hydrogen bonding network at the protein-protein interface is of crucial importance for obtaining reliable binding free energies. The successful reproduction of relative binding energies shows that this type of methodology can be very useful as an aid in rational design and redesign of biologically active macromolecules.     

Calcium binding to the chloroplast andE. coli (CF0) F0 subunit III (c) of the ATP-synthase

Summary Subunit III and c, the 8 kDa components of the chloroplast CF₀, andE. coli H⁺ channel complexes respectively, were isolated and purified for the purpose of studying their Ca⁺⁺-binding properties. Purified subunit III or c as well as the unfractionated organic-solvent soluble preparation from chloroplasts were used in a⁴⁵Ca⁺⁺-ligand blot assay known to detect high affinity Ca⁺⁺-binding sites in proteins. Both subunit III and c showed strong⁴⁵Ca⁺⁺-binding. None of the other CF₀ subunits bound Ca⁺⁺ and of the CF₁ only a weak binding was detected in the region of the , subunits. The Ca⁺⁺-binding was inhibited after treating the proteins in solution by derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La³⁺. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivatizes the hydrophobically located Glu 61 (Asp 61 inE. coli), did not inhibit the Ca⁺⁺-binding in either protein. The data indicate that for both proteins the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III (or c) C-terminal provide some of seven essential oxygen ligands normally required for defining a Ca⁺⁺-binding site in proteins. Based on the accepted models for the hairpin conformation of the subunit III (c), it seems clear that the Ca⁺⁺-binding site can form on the lumenal side of the membrane in the functional CF₀ structures or on the periplasmic side of theE. coli membrane. A working hypothesis we are testing is that Ca⁺⁺-binding to the CF₀ (or F₀) can form an easily reversible gating site such as to enhance the probability for membrane-localized H⁺ gradients being coupled to ATP formation under moderate energization loads, but under excess energization the local H⁺ ion concentration may build up high enough to displace the bound Ca⁺⁺, resulting in delocalization of the H⁺ gradient. The latter situation seems, in chloroplasts at least, to function as a signal for over-energization; i.e., excess light absorption, a potential stress situation for plants. Lumenal acidification appears to be a trigger for initiating stress alleviation responses.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Puschchino, Russia.     

Analysis of the effects of chloride and 2,3-diphosphoglycerate on the cooperative binding of oxygen to hemoglobin.

Analysis of experimental equilibrium constants for the oxygenation of hemoglobin leads to a plausible mechanism for the effect of pH and of chloride ions on cooperativity in hemoglobin. According to this mechanism, the structural changes responsible for cooperativity in chloride- and 2,3-diphosphoglycerate-free hemoglobin are affected only slightly by changes in pH, and the effect of chloride can be accounted for by sequential binding and release of chloride ions during oxygenation.     

Computational study of ligand binding to protein receptors

We have determined, for the first time, the enthalpic contributions to the energy change associated with ligand reorganization (LR) upon the binding of the same ligand to multiple sites within human serum albumine (HSA). Quantum mechanics based density functional theory (DFT) has been used for the LR calculations, which provides much better accuracy than previously used molecular mechanics methods (MM). Our findings show that for some ligands these enthalpic contributions can be attributed to specific structural and conformational changes.