Urease structure

Bacterial catabolism of oral food residue is probably responsible for a higher [NHj] in the oral cavity than in the rest of the respiratory tract.Ammonia, the by-product of oral bacterial protein catabolism and subsequent ureolysis, desorbs from the fluid lining the oral cavity to the airstream.. Saliva, gingival crevicular fluids, and dental plaque supply urea to oral bacteria and may themselves be sites of bacterial NH3 production, based on the presence of urease in each of these materials.Consequently, oral cavity fNTi3)4 is controlled by factors that influence bacterial protein catabolism and ureolysis. Such factors may include the pH of the surface lining fluid, bacterial nutrient sources (food residue on teeth or on buccal surfaces), saliva production, saliva pH, and the effects of oral surface temperature on bacterial metabolism and wall blood flow. The role of teeth, as structures that facilitate bacterial colonization and food entrapment, in augmenting [NH3J4 is unknown. 

The dinuclear active site of urease (1) has been studied in great detail23-29 and has inspired manifold model studies—hence a separate section, Section 6.3.4.12.7, is dedicated to the coordination chemistry related to urease. E. coli Glx I is the first example of a Ni-dependent isomerase and contains a single Ni11 ion coordinated by two histidines, two axial carboxylates of glutamic acid, and two water molecules (2).30-32 It is not active with Zn bound, which is believed to result from the inability of the Zn-substituted enzyme to bind a second aqua ligand and to adopt a six-coordinate structure. 

Biomimetic chemistry of nickel was extensively reviewed.1847,1848 Elaborate complexes have been developed in order to model structural and spectroscopic properties as well as the catalytic function of the biological sites. Biomimetic systems for urease are described in Section 6.3.4.12.7, and model systems for [Ni,Fe]-hydrogenases are collected in Section 6.3.4.12.5. 

Figure 3.21 — (A) Integrated FET with two hydrogen ion-sensitive FET elements. (B) Structure of enzyme-modified FET sensor S plastic card FET enzyme-modified FET chip lUM, immobilized urease membrane. (C) Flowthrough cell Bl fixed sensor cell block B2 movable sensor cell block SC flowthrough cell EC electrical connector RP silicone rubber sheet AMP amplifier. (Reproduced from [151] with permission of Elsevier Science Publishers).

The crystal structure of urease form Klebsiella aerogenes has recently been determined (47). The two nickel(II) ions in the active site are... 

The structure of the urease active center is similar to that of adenosine deaminase, an enzyme containing one zinc(II) per active site (though see 48). This enzyme catalyzes the deamination of adenosine to inosine and NH3 (see Scheme 9), a reaction mechanistically related... 

Jack bean urease is a trimer or hexamer of identical 91-kDa subunits while that of the bacterium Klebsiella has an (a(32y2)2 stoichiometry. Nevertheless, the enzymes are homologous and both contain the same binickel catalytic center (Fig. 16-25).435-4373 The three-dimensional structure of the Klebsiella enzyme revealed that the two nickel ions are bridged by a carbamyl group of a carbamylated lysine. Like ribulose bisphos-phate carboxylase (Fig. 13-10), urease also requires C02 for formation of the active enzyme.438 Formation of the metallocenter also requires four additional proteins, including a chaperonin and a nickel-binding protein.438 439... 

Figure 16-25 The active site of urease showing the two Ni+ ions held by histidine side chains and bridged by a carbamylated lysine (K217 ). Abound urea molecule is shown in green. It has been placed in an open coordination position on one nickel and is shown being attacked for hydrolytic cleavage by a hydroxyl group bound to the other nickel. Based on a structure by Jabri et al.i36 and drawing by Lippard.437...

The first enzyme that was demonstrated to contain nickel was urease (urea amidohydrolase) from jack bean. It catalyzes the hydrolysis of urea to ammonia and carbon dioxide. The protein has a multimeric structure with a relative molecular mass of 590,000 Da. Analysis indicated 12 nickel atoms/mol. Binding studies with the inhibitors indicated an equivalent weight per active site of 105,000, corresponding to 2 nickel atoms/active site. During removal of the metal by treatment with EDTA at pH 3.7, the optical absorption and enzymatic activity correlated with nickel content. This, combined with the sensitivity of the enzyme to the chelating agents acetohydroxamic acid and phos-phoramidate, indicates that nickel is essential to the activity of the enzyme (1). 

In extended X-ray absorption fine structure (EXAFS) studies of urease, Hasnain, Piggott, and co-workers (33, 34) demonstrated that spectra were similar to those of benzimidazole complexes, consistent... 

Second, the correlation of change in enzymic activity with the titration of essential sulfhydryl groups has led to a postulation of eight active sites per 480,000 (56). Unfortunately, the possibility of structural changes during such titrations makes interpretation of such data equivocal. However, the observation that urease retained its activity in 8 M urea, where the molecular weight has been reduced at least to 90,000 (7), supports the conclusion above. 

Third, inhibitor binding studies have led to the conclusion that only two active sites are present in a (16n) structure (94). This conclusion is based on the characterization of a complex containing only 2 moles of hydroxamic acid per (16n) urease and the demonstration that this complex has no catalytic activity. Again, the possibility of structural changes cannot be excluded. 

L. Holm and C. Sanders, An evolutionary treasure unification of a broad set of amidehydrolases related to urease, Protein Structure, Function, and Genetics 1997, 28, 72-82. 

Urease (urea amidohydrolase) is an enzyme first identified over a hundred years ago in bacterial extracts [22], The presence of urease is a virulence factor for some pathogenic bacteria [23,24], It is now known to occur also in plants, fungi, and invertebrates (see [24,25] for reviews). Urease from jack bean was the first enzyme to be crystallized, in 1926. Almost 50 years later its metal content was reexamined and it was found to contain two atoms of nickel per subunit [26]. Finally in 1995 the crystal structure of the enzyme from the enteric bacterium Klebsiella aerogenes was determined [27], Amino-acid sequence comparisons predict that the structures of the plant and bacterial enzymes are similar, although with different subunit arrangements. 

A clearer picture emerged when the crystal structure of K. pneumoniae urease was determined [27], The nickel atoms in the center, Ni-1 and Ni-2, are 3.5 A apart. They are bridged by a carbamyl group, formed from C02 and a lysine residue, explaining the requirement for hydrogen carbonate in reconstitution. The other ligands are two histidines for Ni-1 and an aspartate and two histidines for Ni-2. 

From the crystal structure of urease, Jabri et al. [27] proposed that urea binds through its carbonyl oxygen, whereas the -NH2 hydrons are hydrogen-bonded to residues in the protein (Figure 1). The structure of the site is such that water molecules in the active site do not coordinate optimally to the nickel ions in the substrate-free form. As a result, the binding of urea is favored [40], A loop of polypeptide forms a flap that covers the active site once urea is bound. This flap includes cysteine 319, which had been believed to be catalytically important [41] and is one of the residues proposed to hydrogen-bond to the urea nitrogens. Mutation of this cysteine to alanine leads to decrease, but not necessarily loss, of activity. 

The structure of the complex of urease with urea in the active site is unknown, because the enzyme-substrate intermediate is very short-lived and has not been trapped. Nevertheless, a number of inhibitors of urease that bridge between the nickel atoms are known. Acetohydroxamate is the most studied and binds slowly but with high affinity (K = 4 vaM [25]). Phosphoroamide is also a slowly binding inhibitor. 2-Thioethanol causes the appearance of sulfur-to-nickel... 

Figure 1 Model of the structure of the active site of urease, after Jabri et al. [27], and reaction cycle. (After Refs. 21,161.)...

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