Structural features of the active centre and quaternary structure of inulinase

Abstract

Now many researchers started to pay attention to the fact that not only traditional sorbents and ion exchangers, but also a number of natural components contained in plant and animal organisms have the ability to absorb other organic molecules and ions, enter into complex formation reactions with metal ions, and exhibit catalytic properties. Such natural substances with sorption and ion-exchange properties include aminopolysaccharides (chitin, chitosan), nucleic and ribonucleic acids, saponins, and enzymes. The provided list of substances can be classified as high-molecular compounds with sorption, ligand and catalytic properties. This study presents the results of the investigation of the functional groups of the active centre of inulinase and the features of its quaternary structure. It was established that the enzyme has a quaternary structure, represented by two subunits with Mr 76900 Da and 10140 Da, which have catalytic activity. The imidazole radical of histidine, SH groups and carboxyl groups take part in the formation of the enzyme-substrate complex. The purpose of this study was investigation of the functional groups of the active centre and some features of the quaternary structure of inulinase.

In this study, we used a preparation of inulinase isolated from Aspergillus awamori Ts2250, purified by ion exchange chromatography on columns with diethylaminoethylcellulose (DEAE): [-O-(CH₂)₂-N(C₂H₅)₂]. The homogeneity of the drug was confirmed by gel electrophoresis [24-25]. The catalytic activity of inulinase was determined on the substrate inulin (Spofa, Czech Republic) spectrophotometrically using resorcinol at λ = 540 nm, and the molecular weight was determined by gel chromatography on Sephadex G-200. The presence of electrophilic groups COO^- was determined using the Dixon method [28], and also by IR spectroscopy on a Vertex-70 device (Bruker, Germany) in the frequency range 4000-400 cm^-1.

The active centre of the enzyme includes γ and δ-carboxyl groups of aspartic and glutamic acid residues, respectively. Hydrogen ion H⁺ splits off from the carboxyl group of the glutamine residue of the enzyme and binds to oxygen connecting rings A and B of the substrate. As a result, the oxygen bond with the ring is broken, and the carbon located in position I of ring A forms a carbonium ion, which is stabilized by COO^- group of the aspartic acid residue of the enzyme. An ОН^- ion delivered by a water molecule interacts with carbonium ion, and H⁺ ion of water is fixed in the place of H⁺ ion lost by the glutamic acid residue in inulinase. After this, inulin molecules leave the enzyme, freeing it for subsequent reaction with the substrate. However, this mechanism is not the only one. Histidine imidazole also takes part in the formation of the enzyme-substrate complex. When interacting with inulin, the imidazole group is hydrogen bonded to the oxygen connecting rings A and B of the substrate. There is also an orientation of the COO^- ion of the enzyme ion relative to the resulting carbonium ion in ring A. Subsequently, Н⁺ and ОН^- ions from water molecules are fixed in inulinase in place of H⁺, lost Glu, and OH^- is fixed on the carbonium ion of inulin.