Open AccessX‐Ray Small‐Angle Scattering of Yeast Glyceraldehyde‐3‐phosphate Dehydrogenase as a Function of Saturation with Nicotinamide‐Adenine‐Dinucleotide
Author(s) -
Durchschlag Helmut,
Puchwein Gerd,
Kratky Otto,
Schuster Inge,
Kirschner Kasper
Publication year - 1971
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1971.tb01282.x
Subject(s) - chemistry , scattering , saturation (graph theory) , dehydrogenase , glyceraldehyde 3 phosphate dehydrogenase , analytical chemistry (journal) , crystallography , enzyme , biochemistry , chromatography , physics , optics , mathematics , combinatorics
Structural changes of yeast glyceraldehyde‐3‐phosphate dehydrogenase as a function of saturation with NAD have been measured with the X‐ray small‐angle scattering technique. Samples of yeast glyceraldehyde‐3‐phosphate dehydrogenase were investigated at pH 8.5 and 40° and five different degrees of saturation with the coenzyme NAD (degree of saturation Y = 0, 0.23, 0.46, 0.72, 0.99). Because of the temperature‐sensitivity of the sample, the use of a special flow device became necessary to keep the time of exposure of the samples to 40° and to X‐ray irradiation below 1 h. Apo‐enzyme revealed a contraction in volume depending upon the degree of saturation. Fully saturated the contraction amounts to 7%. As the observed degree of contraction is not linearly proportional to the degree of saturation, the simple sequential mechanism cannot explain the observations. To the extent that they coincide with the quantitative predictions of the allosteric model for this particular enzyme, based on calculations involving the equilibrium constants obtained from chemical relaxation experiments under identical conditions, they strongly support the latter mechanism. Comparison of the experimental scattering curves with theoretical scattering curves of compact full cylinders did not bring about satisfactory agreement in the range of the first submaximum. Hollow circular cylinders with h :2 r 1 = 0.6:1 (apo‐enzyme) or h :2 r 1 = 0.58:1 (holo‐enzyme) may be regarded as sufficiently equivalent in scattering as far as the form of the main maximum is concerned as well as the position and relative intensity of the submaximum. Even if the molecule consists of four subunits they consequently must be arranged in such a way that the entire particle is very similar to a hollow cylinder. The experimental determination of the radius of gyration ( R = 32.1 Å in the case of the apo‐enzyme, or 31.7 Å in the case of the holo‐enzyme) leads to the calculation of the absolute particle dimensions: Apo‐enzyme: h = 48.5 Å, r 1 = 40.4 Å, r 2 = 6.1 Å, Holo‐enzyme: h = 46.4 Å, r 1 = 40.2 Å, r 2 = 6.0 Å. The nature of the structural changes occuring in the allosteric transition can now be described in terms of an increase of the inherent anistropy and of a considerable decrease in the solvent included in the tetrameric assembly. Exposure of the enzyme to 40° over a longer period of time leads to significant alterations in the scattering curves. These can be interpreted on the basis of growing aggregates in polydisperse solutions. This process is accompanied by a considerable decrease of enzyme activity.