Structural change of jack bean urease induced by addition surfactants studied with synchrotron‐radiation small‐angle X‐ray scattering

Abstract

(Received (March 23, 1993) -EJB 93 0417/2Both the quaternary and subunit structures of jack bean urease in solutions with ionic and nonionic surfactants have been studied by small-angle X-ray scattering using a synchrotron-radiation source. The effects of those surfactants on the enzyme activity of urease have also been investigated in the same kind of solvent systems as those used for the scattering experiments. The present results show that the quaternary structure of urease in solution is fairly elongate and that by the relatively minor binding of SDS (SDS/protein = 0.23/1) the native urease molecule is dissociated into six identical subunits with nearly spherical structures. In addition the enzyme activity of urease was mostly retained under every solvent condition investigated, indicating that the subunit found in the present scattering experiments is the fundamental monomeric unit for both the quaternary-structure formation and enzyme function of urease.Jack bean urease, first isolated as a crystalline protein by Sumner [l], is one of the enzyme proteins the catalytic mechanisms and physicochemical properties of which have been studied by many investigators. This enzyme is known as a nickel metalloenzyme with two nickel iondsubunit, where the nickel ion plays an essential role in catalysis [2-51. Previous reports are in agreement with the view that jack bean urease is composed of several subunits equal in shape, however, the molecular mass of the urease molecule is reported in the range 480-590 kDa and that of the subunit in the range 30-97 kDa. The number of the subunits reported also ranges over 4-8 [6-131. Whereas, a recent study using complete amino-acid sequence analysis suggests that the urease subunits adopt a hexameric structure and that a subunit with molecular mass 90770 Da consists of three major domains [14]. In spite of this controversy over the urease structure, there had been no evidence using small-angle X-ray scattering which is known to be a very useful method for studying macromolecular structures in solutions. Consequently, some ambiguity regarding the structure of urease has remained. Therefore, to study the effects of several surfactants on the urease structure we have performed the smallangle X-ray-scattering experiments and clarified both the quaternary and subunit structures of urease in solution. Correspondence to M. Hirai, Department of Physics, Faculty ofEnzyme. Jack bean urease (EC 3.5.1.5).General Studies, Gunma University, Maebashi-shi, Japan, 371 MATERIALS AND METHODS Sample preparation and enzymic activity measurementThe type C-3 urease from jackbeans produced by Sigma Chemical Co. was used as a source of a urease, and further purified by preparative disk electrophoresis (4% polyacrylamide/agarose gel). The fractionated enzyme solution was dialyzed against 0.05 M Hepes, pH 7.0, concentrated using collodion bags, and stored as a stock enzyme solution. This solution is termed native solution or control in the following text and figures. For the preparations of the solutions w...

Temperature- and pressure-dependent stopped-flow kinetic studies of jack bean urease. Implications for the catalytic mechanism

“…Obviously, this means that the pressure did not bring about an irreversible loss of activity due to denaturation. However, this could also mean that (1) the enzyme dissociates into subunits under pressure but rapidly re-associates without denaturation when depressurized, (2) the enzyme dissociates permanently, but the combined activity of the subunits is the same as that of the hexamer, or (3) there is no dissociation of the enzyme into subunits under applied pressure.Importantly, it was shown in studies on the chemical denaturation of urease to half-units [59] and subunits [60] that the quaternary structure of urease is not required for catalytic activity, and—more importantly–that the subunit is not only the fundamental unit for the quaternary structure of urease but also for its activity.The crystal structures of ureases revealed that the active sites are always located in the α subunits of the enzyme, and are entirely independent [26]. This physical independence of the sites thus supports the notion that the monomeric form of jack bean urease should be active.…”

Protein folding at the air–water interface studied with x-ray reflectivity

“…As has recently been determined by small-angle x-ray scattering in aqueous solution (26), the radius of gyration of urease is about 48 Å. These experimental data were successfully modeled (26) assuming that urease hexamers are composed of six cylindrical subunits of 28.7 Å radius and 44.4 Å height. Evidently, the thicknesses of both the glucose oxidase and the urease layers are close to the real protein dimensions in three-dimensional crystals.…”

Novel route for layered double hydroxides preparation by enzymatic decomposition of urea

“…Interestingly, urease is a specific enzyme which catalyzed the hydrolysis of urea [12]. The enzymatic active site is a dinuclear Ni-Ni complexes located in the C-chain of the monomer [13,14]. The bioreaction takes place under mild conditions in a pH range from 4 to 9, according the generation of basic ammonium carbonate:…”