Urease active site studies

Karplus PA, Pearson MA, Hatrsinger RP (1997) 70 Years of crystalUne urease what have we learned . Acc Chem Res 30(8) 330-337. doi 10.1021/ar960022j Leopoldini M, Marino T, Russo N, Toscano M (2008) On the binding mode of urease active site inhibitors a density functional study. Int J Qrrant Chem 108(11) 2023 2029. 

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 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). 

Dixon et al. (35) have proposed a mechanism for urease catalysis (Fig. 3) based on studies of the reactions with the poor substrates formamide, acetamide, and iV-methylurea. They suggest that the two nickel ions are both in the active site, one binding urea and the other a hydroxide ion which acts as an efficient nucleophile. This implies that the nickel ions are within 0.6 nm (1 nm = 10 A) of each other so far it... 

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. 

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... 

Biophysical studies of the urease metal centre in the presence and absence of inhibitors, in conjunction with kinetic data provide the model of the bi-Ni site shown in 1. Certain inhibitors are thought to bridge the two nickel atoms consistent with a bridged transition state during urea hydrolysis. The ligands for nickel are believed not to contain sulphur, however, an essential cysteine is proximal to the active site. Comparisons of diethylpyrocarbonate reactivity for apo- and halo-enzyme are consistent with His as a ligand to nickel (Lee et al., 1990). 

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. 

The X-ray crystallographic studies of the enzyme isolated from Klebsiella aerogenes reveals an active site composed of a dinickel center with 3.5-A Ni- Ni separation. A schematic view of the active site of urease isolated from K. aerogenes is... 

Cr (13.50), and free urea is hydrolyzed slowly in base. Studies with the inert metal centres suggest that coordination through oxygen alone does not activate urea towards hydrolysis. This result suggests that coordination of urea to a single Ni" at the active site of urease is insufficient for catalysis and that a mechanism involving the two metal ions at the active site may be required. 

According to optical, NMR, and X-ray absorption spectroscopic data, the two Ni" sites in truncated UreE are distinct. Both sites are five- or six-coordinate, but they differ in the number of histidine donors. A model in which both metal sites are housed at the dimer interface has been proposed based on site directed mutagenesis studies. " In this model, one Ni" ion is coordinated by four histidines and the other by two histidines. The site with four histidines is postulated to be crucial in the urease activation mechanism. The Cu" substituted UreE also has two distinct metalbinding sites, but optical and resonance Raman data indicate the presence of a cysteine ligand not detected for the Nr loaded protein. "... 

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