Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands

Abstract

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP-pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers cah be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.

Purification and Characterization of Norcoclaurine Synthase

“…The native molecular mass of 28 kDa, determined by gel filtration chromatography, suggests that the native enzyme is a dimer comprised of two 15-kDa subunits. The dimeric structure of NCS was corroborated by the formation of a 28-kDa polypeptide by chemical cross-linking of the native protein complex (15). These results are at variance with the native molecular mass of 15.5 kDa previously reported for NCS (5).…”

“…The void volume was calculated by the elution of blue dextran 2000. The native protein was treated with dimethyl suberimidate as a chemical cross-linking agent as described previously (15). Protein spots from two-dimensional gel electrophoresis gels were analyzed using a PerSeptive Biosystems Voyager DE-STR matrix-assisted laser desorption ionization-time of flight mass spectrometer after in-gel trypsin cleavage.…”

Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

“…The reactions were terminated by adding 200-mM Tris-HCl, pH 8.0. To detect protein cross-linking, each reaction mixture was solubilized by adding an equal volume of Laemmli sample buffer containing 0.1% bromophenol blue, and product contents were ran on 15% SDS PAGE [36]. To validate intermolecular cross-linking further, protein-protein docking was executed using the HEX program [37].…”

“…Protein–protein docking was examined with the HEX program, which is based on a rigid body protein-docking algorithm that explicitly determines the steric shape, electrostatic potential, and charge density of the protein as expansions of spherical polar Fast Fourier Transformation (FFT) basis functions [36]. The surface shapes of each subunit of Wol RNAP (α2ββ'σ) with Wol GreA were calculated to determine their matching potential.…”

Immobilization of Enzymes: An Approach to Fundamental Studies in Biochemistry

“…For example, isopropylrnalate synthetase in the presence of leucine, the feedback inhibitor, exists mainly in dimeric form; in the presence of acetyl-CoA, which competes with leucine kinetically, cross-linked tetramers together with dimers are obtained (57). The character of the intersubunit linkage changes in pyruvate kinase, in the presence of ADP or fructose-l,6-diphosphate (65), and for phospholipase-A2 in the presnece of phospholipids and metal cations (236). For phosphofructokinase (68) and creatine kinase (146), a correlation was established with the help of immobilization between the quaternary structure of enzymes, activity, and allosteric properties.…”