Urease active site structure

Remaut H, Saeaeov N, Ciueli S and Van Beeu-MEN J (2001) Structural basis for Ni transport and assembly of the urease active site by the metal-lochaperone UreEfrom Bacillus pasteurii. J Biol Chem 276 49365-49370. 

A for the two histidines. The other two Cu" ions are found in surface exposed sites coordinated by two histidine e nitrogens and one or two water molecules. The putative cysteine ligand identified by spectroscopy and mutagenesis " does not bind Cu" in the structure and is quite distant from the metal-binding sites. It may be that this interaction occurs in solution between a cysteine residue from one dimer and a Cu" ion from a second dimer, and is precluded in the structure by crystal packing. The dimer interface site is proposed to deliver Ni" ions one at a time to the urease active site, and the other two sites are proposed to play a more secondary role, serving as reservoirs for Ni". ... 

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. 

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... 

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). 

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. 

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.)...

Roughly 30% of enzymes are metalloenzymes or require metal ions for activity and the present chapter will concentrate on the chemisty and structure of the plant metalloenzymes. As analytical methods have improved it has been possible to establish a metal ion requirement for a variety of enzymes which were initially considered to be pure proteins. A dramatic example is provided by the enzyme urease isolated from Jack beans and first crystallised by Sumner (1926) (the first enzyme to be crystallised). Sumner defined an enzyme as a pure protein with catalytic activity, however, Zerner and his coworkers (Dixon et al., 1975) established that urease is in fact a nickel metalloenzyme. Jack bean urease contains two moles of nickel(II) per mole of active sites and at least one of these metal ions is implicated in its mechanism of action. 

Metalloenzymes pose a particular problem to both experimentalists and modelers. Crystal structures of metalloenzymes typically reveal only one state of the active site and the state obtained frequently depends on the crystallization conditions. In some cases, states probably not relevant to any aspect of the mechanism have been obtained, and in many cases it may not be possible to obtain states of interest, simply because they are too reactive. This is where molecular modeling can make a unique contribution and a recent study of urease provides a good example of what can be achieved119 1. A molecular mechanics study of urease as crystallized revealed that a water molecule was probably missing from the refined crystal structure. A conformational search of the active site geometry with the natural substrate, urea, bound led to the determination of a consensus binding model[I91]. Clearly, the urea complex cannot be crystallized because of the rate at which the urea is broken down to ammonia and, therefore, modeling approaches such as this represent a real contribution to the study of metalloenzymes. 

Historically, the first chemical synthesis of urea by Wohler, from ammonium cyanate in 1828, was a milestone that established a bridge between inorganic and organic chemistry. Urease was the first enzyme ever to be crystallized (6), and it was the first protein shown to contain nickel ions in the active site (7). The first X-ray crystal structure of urease became known in 1995 (8). Significant progress was made since then toward an understanding of its catalytic mechanism, as well as toward the structural and functional emulation of its active site by synthetic model complexes (5, 9). 

The structure of urease from BPU was solved also with p-mercaptoethanol bound at the active site (134), where the inhibitor symmetrically bridges the two nickel ions through its sulfur atom (J(Ni Ni) = 3.1 A) and chelates one nickel through the terminal OH functionality, akin to the mode of inhibition by AHA. Because of the bridging thiolate, the two nickel ions are strongly antiferromagnet-ically coupled (23). No synthetic model for the p-mercaptoethanol-inhibited active site was reported yet. 

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