Mechanism of urease

The results of most model studies for Ni-mediated urea degradation reported to date are consistent with a cyanate intermediate. While this differs from the most likely mechanism of urease activity as deduced from protein crystallography, there is still no definitive evidence ruling out a transient Ni-bound cyanate intermediate for the enzyme. 

Figure 15.2 Reaction mechanism of urease. Ni 1 binds urea and acts as a Lewis acid to polarise the carbonyl group, making its carbon more electrophilic, while Ni 2 facilitates deprotonation of a bound water molecule to generate a nucleophilic hydroxyl species. (From Ragsdale, 1998. Copyright 1998, with permission from Elsevier.)...

The mechanism of urease action is probably related to those of metalloproteases such as carboxypeptidase A (Fig. 12-16) and of the zinc-dependent carbonic... 

The initially proposed mechanism of urease activity (Scheme 1) assumed that urea is first polarized and activated by monodentate O-coordination to one Ni(II) ion, in conjunction with extensive hydrogen bonding within the active site pocket of the protein. The retained water on Ni2 becomes deprotonated, while a nearby histidine residue becomes protonated. The resulting Ni2-bound terminal hydroxide then acts as the nucleophile and attacks the carbonyl C of urea. The reaction proceeds through a tetrahedral intermediate, from which NH3 is released, assisted... 

Figure 2 Mechanism of urease. This proposed mechanism features binding of urea through its carbonyl group to one of the Ni ions, making the carbon subject to nucleophilic attack by the bridging hydroxide leading to liberation of NH2, which is protonated by to form ammonia,...

Figure 2 Mechanism of urease. This proposed mechanism features binding of urea through its carbonyl group to one of the Ni ions, making the carbon subject to nucleophilic attack by the bridging hydroxide leading to liberation of NH2, which is protonated by to form ammonia, and carbamate, which hydrolyzes to another mole of ammonia and carbonic acid. The same mechanism with proton transfer from water (instead of histidine) to ammonia is another possibility. Another possibility would be urea binding in a monodentate manner with a terminal hydroxide on Ni2 as the nucleophile...

Figure 15.4(A) shows the effect of the R = Zn2+/Al3+ ratio, which determines the charge density of the LDH layer, on the Freundlich adsorption isotherms. K values are far higher than those measured for smectite or other inorganic matrices. The increase in Kf with the charge density (Kf= 215, 228, 325mg/g, respectively, for R = 4, 3 and 2) is supported by a mechanism of adsorption based on an anion exchange reaction. The desorption isotherms confirm that urease is chemically adsorbed by the LDH surface. The aggregation of the LDH platelets can affect noticeably their adsorption capacity for enzymes and the preparation of LDH adsorbant appears to be a determinant step for the immobilization efficiency. [ZnRAl]-urease hybrid LDH was also prepared by coprecipitation with R = 2, 3 and 4 and Q= urease/ZnRAl from 1 /3 up to 2.5. For Q < 1.0,100 % of the urease is retained by the LDH matrix whatever the R value while for higher Q values an increase in the enzyme/LDH weight ratio leads to a decrease in the percentage of the immobilized amount.

Kinetic evidence obtained for intramolecular proton transfer between nickel and coordinated thiolate, in a tetrahedral complex containing the bulky triphos ligand (Pl PCE CE PPh to prevent interference from binuclear p-thiolate species, is important with respect to the mechanisms of action of a number of metalloenzymes, of nickel (cf. urease, Section VII. B.4) and of other metals (289). 

Kinetics of formation of the dinuclear iron(III) complex [(tpa)Fe (p-0)(p-urea)Fe(tpa)]s+ tpa = tris(2-pyridylmethyl)amine were investigated in relation to the suggestion that urease action in vivo involves an intermediate containing Ni (p - O H) (p -ur e a) Ni. The mechanism of formation of the di-iron species from [(tpa)(H20)Fe(p-0)Fe(0H)(tpa)]3+ is proposed to involve three reversible steps (350). Three kinetically distinct steps are also involved in the deposition of FeO(OH) in... 

Given that hydroxylamine reacts rapidly with heme proteins and other oxidants to produce NO [53], the hydrolysis of hydroxyurea to hydroxylamine also provides an alternative mechanism of NO formation from hydroxyurea, potentially compatible with the observed clinical increases in NO metabolites during hydroxyurea therapy. Incubation of hydroxyurea with human blood in the presence of urease results in the formation of HbNO [122]. This reaction also produces metHb and the NO metabolites nitrite and nitrate and time course studies show that the HbNO forms quickly and reaches a peak after 15 min [122]. Consistent with earlier reports, the incubation ofhy-droxyurea (10 mM) and blood in the absence of urease or with heat-denatured urease fails to produce HbNO over 2 h and suggests that HbNO formation occurs through the reactions of hemoglobin and hydroxylamine, formed by the urease-mediated hydrolysis of hydroxyurea [122]. Significantly, these results confirm that the kinetics of HbNO formation from the direct reactions of hydroxyurea with any blood component occur too slowly to account for the observed in vivo increase in HbNO and focus future work on the hydrolytic metabolism of hydroxyurea. 

Fig. 9. A possible mechanism for urea hydrolysis at the catalytic site of urease.

A possible mechanism for the action of urease is pictured in Fig. 16-25 and Eq. 16-47. Carbamate is thought to be one intermediate. Can you suggest an alternative possibility for the initial nickel ion-dependent steps. See Barrios and Lippard.476... 

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... 

The mechanism involved in the production of urease oligomers on gel columns has not been clarified and seems anomalous because all of the species were excluded from the gel. Moreover, as Creeth and Nichol observed (39), if an equilibrium exists, the kinetic constants are small. 

The Enzyme Commission catalog (EC 3.5.1.5) lists the urease reaction as urea + 2 H20 = C02 + 2 NH3. Since two C-N bonds are broken it is evident that the stoichiometric relation above is the result of two component reactions. Any conjecture concerning the mechanisms of these reactions and the nature of the intermediates must encompass the action of inhibitors and the spectrum of substrates. Some of the organic inhibitors that have been reported are shown in Table I. The substrates that have been shown to be hydrolyzed are listed in Table II. 

Until our knoweldge of the ureases discloses biological significance the research on these enzymes will continue to be restricted. Inquiries into the mechanism of the urease catalysis are clearly appropriate... 

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. 

Dixon et al. (1980) have proposed a mechanism for the action of urease (Fig. 5-3). The mechanism involves nucleophilic attack by hydroxide coordinated to one nickel, on urea which is coordinated to the other nickel(II) via the carbonyl oxygen. 

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). 

In developing some of the elementary principles of the kinetics of enzyme reactions, we shall discuss an enzymatic reaction that has been suggested by Levine and LaCourse as part of a system that would reduce the size of an artificial kidney. The desired result is the production of an artificial kidney that could be worn by the patient and would incorporate a replaceable unit for the elimination of tte nitrogenous waste products such as uric acid and creatinine, In the microencapsulation scheme proposed by Levine and LaCourse, the enzyme urease would be used in tire removal of urea from ti)e bloodstream. Here, the catalytic action of urease would cause urea to decompose into ammonia and carbon dioxide. The mechanism of the reaction is believed to proceed by the following sequence of elementary reactions ... 

Fig. 26 Cross-sectional view of a bioerosion-regulated hydrocortisone delivery system, a feedback-regulated drug delivery system, showing the drug-dispersed monolithic bioerodible polymer matrix with surface-immobilized ureases. The mechanism of release and time course for the urea-activated release of hydrocortisone are also shown. (From Ref > 1)...

The maximal intestinal immunization can be achieved by intra-Peyer s patch immunization, and thus this method can be used to screen oral vaccine candidate antigens without the added complication of simultaneously testing oral-delivery systems. Immunization of subjects against Helicobacter pylori by intra-Peyer s patch resulted in an 84-91% reduction in H. pylori infection compared with unimmrmized controls. The therapeutic efficacy of the recombinant H. pylori urease vaccine in mice was shown to be comparable with that achieved with the combined anti-biotic/antacid treatment in humans. The oral vaccination is preferred to conventional treatment of ulcers because it is a very simple and quick procedure compared with long-term conventional treatment. In addition, vaccines use the defense mechanisms of the body to establish long-lasting immunity. ... 

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