Hasko H. Paradies

Phosphofructokinase: Structure and Function

Publisher Summary This chapter explores the structure and function of phosphofructokinase. Phosphofructokinase is a well-known regulatory enzyme responsible for the control of the glucose flux through the glycolytic pathway. The reaction catalyzed by this enzyme is relatively simple, but it is complicated by a variety of modulators that affect the rates of reaction. The chapter describes the molecular structure of phosphofructokinase from several sources and relates them to the function of the molecule. Several groups have carried out kinetic studies upon the enzyme in an attempt to understand the action of the various effectors. Such studies taken in conjunction with ligand-binding experiments have resulted in a possible model for the regulation of enzyme activity. These processes may be studied on a microscopic level by employing a variety of physical techniques such as low-angle X-ray scattering, analytical ultracentrifugation, and inelastic and elastic light scattering, in addition to various spectroscopic methods.

The structure of the δ-subunit from chloroplast coupling factor (CF1) in solution

Abstract The δ-subunit from chloroplast coupling factor (CF 1 ) was isolated according to Younis et al. (1977 : J. Biol. Chem. 252: 1814–1818) and further purified by means of DEAE-cellulose chromatography in the absence of Mg 2+ at pH 8.0, but in the presence of 5 % polyethylene-glycol. The homogeneous δ-protein fraction was studied by inelastic light scattering measurements, small angle X-ray scattering experiments and analytical ultracentrifugation. The hydrodynamic measurements and the small angle X-ray scattering experiments in solution support the hydrodynamic description of a prolate ellipsoid of revolution with gross dimensions of 2a = 25.0 A, 2b = 28.0 A and 2c = 90.0 A, having a radius of gyration of 21.80 A, a sedimentation coefficient of 1.70S and a translational diffusion constant of D = 3.92 × 10 7 cm 2 · sec −1 . Considering different molecular conformations, e.g. worm-like coil, ellipsoid of revolution, and flexible rod, the description of a flexible rod is the most probable one, when one considers the hydrodynamic values obtained for the δ-subunit.

Size and shape of nonionic micelles determined by x-ray scattering techniques

Abstract Small angle X-ray scattering studies on aqueous solutions of the nonionic detergents Triton X-100, Lubrol WX, and two other n -alkyl polyoxyethylene ethers, in the concentration range of 5–15 mg/ml were performed and their size, degree of hydration and radii of gyration were determined. By densimetric methods the partial specific volumes were determined and compared with the values obtained from X-ray scattering techniques at different contrast. The radii of gyration for the nonionic detergents in aqueous solutions at T = 293°K were found to be in the range of R g = 29.5–34.5 A, their volumes in the range of 3.35–4.15 × 10 5 A 3 and their degree of hydration about 1.15 g H 2 O/g micelle. However, the inverted micelle of Triton X-100 in hexane, containing 0.03–0.05% (w/w) water, has a radius of gyration of R g = 10.2 A. The behavior of the hydrophobic part of the Triton X-100 micelles In aqueous and nonaqueous solutions was studied with tag-labelled iodine, introduced into the phenyl ring, by means of X-ray scattering at various contrast. Assuming a two-level step function in electron density within the Triton X-100 micelles, the high electron density regions extend to R = 25.7 A ( T = 293°K), determined from the iodine labelled phenyl ring. From the correlation function, calculated directly from the scattering data, the hydrocarbon core of the tag-labelled Triton X-100 yields a maximum dimension of 48.0 A. The value of the average curvature of the tag-labelled hydrophobic core of Triton X-100 was determined to 1/23.6 A, smaller than the average radius calculated from the radius of gyration for the hydrophobic part. This shows that the core surface is convoluted, e.g., some oxyethylene chains must be embedded in the hydrophobic core, indicating that there is no direct sharp boundary between the hydrophobic interior and the oxyethylene units. Whereas the largest dimensions of the Triton X-100 micelles as well as of Lubrol WX are 107–110 A at T = 293°K, determined from the correlation function, the extension in solution of the inverted micelle of Triton X-100 was found to be 26.5 A. Density homogeneities, namely variations of the average density of the Triton X-100 particles, can introduce anomalies in I (O, p o ) and R g 2 ( p o ) close to buoyancy and the critical point. The data suggest that between 20–50°C the scattering data in 0.01 M TRIS-HCl are influenced by internal density fluctuations, e.g., entrapping of water, rather than micelle size.

Effect of ATP on the translational diffusion coefficient of the α-subunit of Escherichia coli F1-ATPase

(ii) One portion, namely the coupling factor (Fr), appears to be peripheral to the membrane without disruption of the phospholipid bilayer, having mol. wt 325 000-370 000 (CFr, ECFr, MFr and TFr) [3-61; The remaining portion of the complex (F,) is an integral part of the membrane and mediates proton translocation. This part of the complex (F,) has mol. wt of an order of 90 000 [7,8], e.g., for CFr F,, and 110 000 for ECFr * F, in detergent solution. Reconstruction experiments for ECFr have been performed revealing that (Y, /3 and 7 subunits are required for ATPase activity [9,10]. Moreover, ATPase activities were reconstituted from complexes containing different subunit proportions of E. coli ATPase [ 1 l] as well as from purified individual subunits of the ATPase of a thermophilic bacterium (TF,) [12]. An important finding obtained through the study of isolated subunits is that (Y binds ATP with a Kd value of 0.9 PM, and 0.1 E.~M for ADP [13]. This paper describes the hydrodynamic property of the o-subunit of ECF, in the presence and absence of ATP by means of inelastic light scattering, indicating

Crystallographic study of single crystals of mitochondrial coupling factor (BF1) from beef heart

Abstract Beef heart mitochondrial coupling factor (BF 1 ) was crystallized from 0.1 M Tris-PO 4 , pH 7.8, containing 1 mM EDTA, 4 mM ATP and 1.85 M (NH 4 ) 2 SO 4 , at 22°C. Single crystals were obtained different from those reported by Spitzberg and Haworth (Biochim. Biophys. Acta 492, 237–240, 1977). The X-ray diffraction pattern reveals an orthorhombic lattice with a = 150 A, b = 132 A and c = 180 A and, according to the absence of reflection, a space group of C222 1 . The crystal density was determined to 1.19 g·ml −1 and, assuming a molecular weight of 350,000 for BF 1 , there is only one half of the BF 1 molecule in the asymmetric unit cell.

On the quaternary structure of native rabbit muscle phosphofructokinase

Abstract Small angle X-ray scattering measurements on solutions of native rabbit muscle phosphofructokinase (EC 2.7.1.11; ATP; D-fructose-6-phosphate 1 phosphotransferase) show that the dimer has a radius of gyration of 32.5 A and a molecular weight of 160,000, and that the biologically active tetramer has a radius of gyration of 51.5 A and a molecular weight of 320.000. A possible model was calculated from scattering curves of the dimer and tetramer suggesting two hollow cylinders with cell dimensions for the dimer of a height of 78.0 A and a long half axis of 38.0 A, and for the tetramer of a height of 155.0 A and an outer radius of 35.0 A. The tetramer is formed along the 78.0 A axis of the dimer by means of an end-to-end aggregation. The overall particle dimensions of the protomer of molecular weight 80,000 is calculated to be 35.0 × 30.0 × 55.0 A, assuming an elliptical molecule. The distance between the centers of the two dimeric units within the tetramer is 104.5 ± 1.5 A.

Molecular properties of the ATP synthetase from Escherichiacoli

Abstract The molecular weight, radius of gyration and volume of the ATP synthetase from Escherichia coli were determined by small angle X-ray scattering in solution. A weight average molecular weight of 448,000 ± 23,000 for the F1·F0-complex was determined by small angle X-ray and laser light scattering at pH 7.8 at 4°C and a molecular weight of 930,000 ± 13,500 was found at 20°C. The maximum cord length of the F1·F0-complex at 4°C was determined to 17.5 ± 1.0 nm and 35.0 ± 2.0 nm at 20°C for the dimer, indicating a linear arrangement of the two F1·F0 monomers within the dimer. A possible hydrodynamic description of the F1·F0 monomer is that of a spherical cone with dimensions of H = 17.0 nm in height, a larger spherical radius of R1 = 8.4 nm and a smaller one of R2 = 5.9 nm, which match the experimental scattering curve satisfactorily.

The structure of the ε-subunit from the chloroplast coupling factor (CF1) studied by means of small angle X-ray scattering and inelastic light scattering

The e-subunit from the chloroplast coupling factor (CF1) was purified on a BioGel A 0.5 m column, and the size, shape and radius of gyration were determined. The diffusion coefficient, D20, wo, was determined by means of inelastic light scattering and was found to be (11.3 ± 0.05) × 10−7 cm2·sec−1 and independent of pH and ionic strength. From the sedimentation coefficient (1.70S) and D20, wo we obtained a molecular weight of 11,900 with a partial specific volume of 0.740 ± 0.003 ml · g−1. The radius of gyration of e in 0.05 M TRIS-HCl, pH 7.0, was found to be 11.8 ± 0.04 A, the volume 17.0 × 103 (A3) and the specific inner surface 0.19 A−1, indicating a spherical molecule with overall dimensions of D = 31.8 A with a given axial ratio of 1:1:2.4. The description of the e-subunit in solution as a prolate ellipsoid of revolution with half axes a = b = 12.7 A and c = 25.4 A was obtained from a comparison of the theoretical and the experimental scattering curves. The degree of hydration was determined to be 0.15 g H2O/g protein.

Two enzymatically active forms of valyl-tRNA-synthetase from E. coli

Abstract This paper reports the purification, kinetic properties and molecular weights of two active forms of valyl-tRNA synthetase from E. coli. Whereas form I catalyses the valine dependent ATP-[ 32 P] pyrophosphate exchange reaction as well as the esterification of tRNA I Val , form II has a different K m -value for the aminoacylation but still catalyses the pyrophosphate exchange reaction with the same K m -value as form I. While form II, with a molecular weight of 125,000, can be dissociated into two unequal parts with molecular weights of M I = 69,000 and M II = 46,500, respectively, form I consists of one polypeptide chain with a molecular weight of 115,000. The data obtained from amino acid analysis indicate that the two fragments also differ in their electrostatic charge capacity.

Inelastic light scattering studies of solutions of the chloroplast coupling factor (CF1) at high and low ionic strength

The translational diffusion coefficient of CF1 at low and high protein concentration as well as at different ionic strength (0.05 – 1.65 M) wsa determined by means of quasi-elastic light scattering experiments. The diffusion coefficient changes from D20,wo = 3.12 × 10−7 cm2 · sec−1 at 0.05 M, pH 7.8, 20°C, to D20,wo = 3.52 × 10−7 cm2 · sec−1 at 1.6 M, pH 7.8, 20°C. At high enzyme concentration (20 mg/ml) and under crystallization conditions (Paradies, BBRC 91: 685, 1979) CF1 behaves as a solution of “true” hard spheres, whereas at low salt concentration the ionic atmosphere has a larger spatial extent, resulting in a higher effective hydrodynamic radius (RH = 65 A).