Urease, carboxylation

Results from an array of methods, including X-ray absorption, EXAFS, esr and magnetic circular dichroism, suggest that in all ureases the active sites are a pair of Ni" atoms. In at least one urease,these are 350 pm apart and are bridged by a carboxylate group. One nickel is attached to 2 N atoms with a fourth site probably used for binding to urea. The second nickel has a trigonal bipyramidal coordination sphere. 

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. 

Fig. 3. Proposed reaction cycle for urease. For urea, R = —NH2. Step 1 urea is activated toward nucleophilic attack by O coordination to a nickel ion the =N+H2 is stabilized by interaction with a protein carboxylate. Step 2 nucleophilic attack by a hydroxide ion, coordinated to the second nickel, to form a tetrahedral intermediate. Step 3 breakdown of the tetrahedral intermediate to form a coordinated carbamate ion. Step 4 hydrolysis releases carbamate ion, the initial product of urease on urea. Reproduced, with permission, from Ref. 34.

Because the urea-urease interaction leads to a pH increase, a polymer that increases erosion rate with increasing pH is needed. A useful polymer for this application is a partially esterified copolymer of methyl vinyl ether and maleic anhydride. This copolymer undergoes surface erosion with an erosion rate that is extraordinarily pH-dependent (J). The polymer dissolves by ionization of the carboxylic acid groups as shown below ... 

Specific active site stmctural features of urease are the bimetalhc arrangement with a Ni Ni distance of 3.5-3.7 A and nonsymmetric N/O-rich coordination environment, the bridging carbamate (often modeled by a bridging carboxylate), and the presence of a hydrolytically active Ni-bound hydroxide or water. Relevant dinickel complexes that emulate at least part of these features are introduced in this chapter. Of course, the ability to bind urea is a prerequisite for urease-like activity, and different urea-binding modes were observed in synthetic model compounds. Those model complexes and artificial systems that mediate the decomposition of urea are discussed in Section III. 

Parallel to the phenolato-based systems, related alkoxo-bridged ligands were developed for emulating the urease active site, in particular by the groups of Krebs, Nakao, and Yamaguchi. In contrast to the related 3 compound, complexes 11 feature only a single carboxylate bridge, but an additional solvent molecule is attached to each metal ion (63, 64). 

Three Ni-confaintng enzymes (see Nickel Enzymes Cofactors) appear to utilize Ni metallochaperones for enzyme activation. UreE appears to function in NP+ delivery to urease. The Klebsiella aerogenes protein binds 6 Ni per dimer, whereas that from Bacillus pasteurii binds a single Ni per dimer. The metal content differences arise from a His-Asp-His sequence near the middle and a histidine-rich region at the carboxyl terminus of the former protein. Truncated K aerogenes UreE protein, missing the His-rich... 

Many other similar applications have been reported such as the electrochemical determination of electroinactive cationic medicines,313 determination of urea,314 uric acid,315 and application to glucose biosensors to decrease interference of ascorbate, urate, and acetaminophen.316 Enzyme immobilized membranes are also sensing membranes, e.g. urea responsive membranes, poly(carboxylic acid) membranes in which urease is immobilized,317 fructose responsive membranes, and polyion complex membranes in which fructose dehydrogenase is immobilized.318 Such applications will expand further in the future and contribute to human life. 

K. Ishihana, N. Muramoto, H. Fujii and I. Shinohara, Preparation and permeability of urea-responsive polymer membrane consisting of immobilized urease and poly-(aromatic carboxylic acid), J. Polym. Sci., Polym. Lett. Ed., 1985, 23, 531-535. 

Several structures of ureases are available (2). In all cases, the active site contains two Ni(II) ions bridged by the carboxylate group of a carbamylated lysine and by a hydroxide ion (Fig. lA). Each Ni is also coordinated by two histidines and one water molecule, whereas Ni(2) is further bound to an aspartate, resulting in a pentacoordinate Ni(l) and hexacoordinate Ni(2). In the resting state of the enzyme from Bacillus pasteurii, the active site accommodates a fourth water molecule, completing a tetrahedral cluster of solvent molecules (12). The access to the active site is regulated by a flexible helix-loop-helix motif, the position of other amino acids involved in the catalysis being also critically affected by the flap movement. 

Ligand and complex design in biomimetic systems is diverse but a few general concepts are normally followed (i) the metal ions employed are often the same as in the native systems e.g. Ni(II) in urease models [68] and (ii) pyridine or pyrazole residues are often used to mimic the histidine residues in the enzymes phenol, carboxylate, pyrazolate or water molecules serve as mimics for bridging residues like aspartate, lysine or water/hydroxide and (iii) dinucleating ligands are used to bring the two metal ions into close proximity. 

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