Urease mechanism

However, details of this process including the mode of urea binding, the protonation state of individual surround protein residues, and the exact identity of the nucleophile are still under debate. Cyanate also was proposed as a possible intermediate in the urease mechanism (33). Recent quantum chemical calculations and molecular dynamics simulations indicated that hydrolytic and ehmination mechanisms might indeed compete, and that both are viable reaction channels for urease (34—37). Finally, an important issue is Why does urease require nickel as the metal of choice, whereas most other metallohydrolases use zinc While it was speculated that, inter alia, the relatively rigid and stable coordination environment around the Ni(II) ions as opposed to the higher kinetic lability and lower thermodynamic stability of Zn(II) complexes might play a role (31), this fundamental question has not yet been answered. 

Scheme 16. Transformations at the dinickel(II) site of 24 relevant to the urease mechanism (72).

In all the above cases, however, subsequent hydrolysis of the formed cyanate could not be achieved, even when the more stable pyrazolate complex with triazacyclononane compartments was used to allow forcing conditions in aqueous solution. Conversions at the bimetallic scaffold of 24 relevant to the urease mechanism are summarized in Scheme 16 (72). 

The uncatalyzed reaction has an activation energy of 125 kJ/mol. The enz Tne catalyzes a mechanism that has = 46 kJ/mol. By what factor does urease increase the rate of urea conversion at 21 °C Hint ... 

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 15.4(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. 

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 formation and excretion of urea is the primary mechanism by which excess nitrogen, in the form of ammonia, is removed from the body. Surprisingly, it was found that the actual rate of urea synthesis exceeded considerably the rate of excretion of the urea. The interesting question, therefore, is what is the fate of this lost urea The answer is that urea enters the large intestine, where it is degraded by microorganisms that possess the enzyme urease, which catalyses the reaction ... 

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

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... 

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... 

Fig. 6. Biochemo-mechanically controlled protein release using urease-loaded gel. The experiment was carried out in a 0.2 M ammonium buffer maintained at 35 °C using insulin (Mw = 5733) as the protein solute. (E. Kokufuta, S. Matsukawa, T. Ebihara, and K. Mat-suda [77])...

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. 

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