The structure of jack bean urease

Abstract

The amino acid sequence of jack bean urease has been determined. The protein consists of a single kind of polypeptide chain containing 840 amino acid residues. The subunit relative molecular mass calculated from the sequence is 90770, indicating that urease is composed of six subunits. Out of 25 histidine residues in urease, 13 were crowded in the region between residues 479 and 607, suggesting that this region may contain the nickelbinding site. Limited tryptic digestion cleaved urease at two sites, Lys-128 and Lys-662. Proteolytic products were not dissociated and retained full enzymatic activity. Five tryptic peptides containing the reactive cysteine residues were isolated and characterized with the aid of sulfhydryl-specific reagents, N-iodoacetyl-N'-(5-sulfo-lnaphthy1)ethylenediamine and N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide. The reactive cysteine residues were located at positions 59,207, 592,663, and 824. The possibility that Cys-59, Cys-207, Cys-663, and Cys-824 are involved in the urease activity of the enzyme has been eliminated. Cys-592, which is essential for enzymatic activity, is located in the above-mentioned histidine-rich region.Jack bean urease was the first enzyme isolated as a crystalline protein by Sumner in 1926 [l]. However, the subunit structure of this enzyme has been somewhat ambiguous. The relative molecular mass reported first for the native urease was 480000 [2] while recently the value of 590 000 was reported [3]. The values reported for the monomer range over 30000-97000 [3-61. On the other hand, the subunits of ureases from microorganisms appear to be smaller than jack bean urease in size and number [7-91. Recently it was suggested that one large and two small polypeptides may be generally associated with microbial ureases [lo].Jack bean urease is also the first example of a nickel metalloenzyme and contains two nickel ions per 96600-M, subunit [ l l , 121. Spectrophotometric studies showed that the nickel ion has an essential role in catalysis and that the substrate and inhibitors of urease bind to the nickel ion [13, 141. A model for the mechanism of action of urease, taking into consideration the nickel ion, has been proposed [15].The presence of sulfhydryl groups in a protein was first described for jack bean urease [16]. The sulfhydryl groups in the enzyme are classified into the exposed ones which are titrated in the native conformation and the buried ones which can be titrated only in the presence of denaturant. Urease has been reported to have 27 -35 reactive exposed cysteine residues per urease molecule [17 -191. These cysteine residues were divided into the inessential ones, which readily react with sulfhydryl reagents like 5,5'-dithiobis(2-nitrobenzoic acid) without activity loss, and the essential ones, which react more

Chemical Cross-linking and Mass Spectrometric Identification of Sites of Interaction for UreD, UreF, and Urease

“…For UreA, four peptides (10,12,19, and 25 residues) were identified that cover 66 of the total 100 amino acids, accounting for 66% of the sequence. Nine UreB peptides (6,7,10,11,12,19,23,23, and 27 residues) were represented, covering 91 of the 106 amino acids and accounting for 86% of the sequence. In 2 See prospector.ucsf.edu.…”

“…Urease sequences from diverse sources are more than 60% identical despite having differences in the numbers of subunits. Plant and fungal ureases contain a single type of subunit, such as the 840-residue polypeptide from jack bean (7). In contrast, Helicobacter pylori urease is comprised of two subunits containing 238 and 569 residues that match distinct portions of the eukaryote enzyme (8).…”

Electrosynthesized MIPs for transferrin: Plastibodies or nano-filters?

“…1B) that assumes surface-target binding as prerequisite of the following EP step for MIP formation. Urease shows the most effective blocking of the gold surface (indicative of the strongest binding to the gold, which is assumed to be due to its cysteine-rich domain (Takishima et al, 1988)), followed in order by HSA, Trf and ferritin.…”

“…Since urease has a much larger molecular weight than Trf, to explain this behavior the molecular structure must be taken into consideration. Urease is a homohexamer (Takishima et al, 1988) and each monomer consists of 4 domains with a molecular weight of 90 kDa (Fig. 6).…”

Urease Mediated Synthesis of Crystalline Porous Tin Oxide at Room-temperature and Applications in Lithium-ion Batteries and Gas Sensors

“…For the first time, the nanoporous structures for non-biological oxides are reported by the direction of bio-molecule at room temperature. The jack bean urease is composed of six 90 KDa subunits (840 residues), and has an isoelectric point of 4.9 [11] . Besides its biocatalytic property for decomposing urea, the jack bean urease also have high affinity for metal ions due to its abundant surface chemical moieties [12] .…”