Urease kinetics

The first applications of enzymes in bioanalytical chemistry can be dated back to the middle of nineteenth century, and they were also used for design of first biosensors. These enzymes, which have proved particularly useful in development of biosensors, are able to stabilize the transition state between substrate and its products at the active sites. Enzymes are classified regarding their functions, and the classes of enzymes are relevant to different types of biosensors. The increase in reaction rate that occurs in enzyme-catalyzed reactions may range from several up to e.g. 13 orders of magnitude observed for hydrolysis of urea in the presence of urease. Kinetic properties of enzymes are most commonly expressed by Michaelis constant Ku that corresponds to concentration of substrate required to achieve half of the maximum rate of enzyme-catalyzed reaction. When enzyme is saturated, the reaction rate depends only on the turnover number, i.e., number of substrate molecules reacting per second. 

Example Applications. Previous work has mostly been concerned with testing the theoretical models and obtaining proof of concept (29,39). Antimony pH tips were used to image the activity of urease immobilized in a disk of glutaraldehyde/BSA gel and to quantify the total flux of H+ in the presence of a saturating concentration of urea (29). Wei et al. (39) showed that urease kinetics can also be quantified using an ammonium-selective neutral carrier-based tip to determine the concentration profile of NHj produced by the hydrolytic reaction... 

Following this procedure urea can be determined with a linear calibration graph from 0.143 p.g-ml To 1.43 p.g-ml and a detection limit of 0.04 p.g-ml based on 3o criterion. Results show precision, as well as a satisfactory analytical recovery. The selectivity of the kinetic method itself is improved due to the great specificity that urease has for urea. There were no significant interferences in urea determination among the various substances tested. Method was applied for the determination of urea in semm. 

Jackbean urease was immobilized on kaolinite and montmorillonite [98]. The amounts of urease required for maximum immobilization were 70 and 90 mg g 1 of kaolinite and montmorillonite, respectively. The Km values of immobilized urease (25.1-60.8 mM) were of the same order of magnitude as that of free urease (29.4 mM) but one order of magnitude higher than those of soil urease (1.77-2.90 mM). Immobilization of urease on clay surfaces leads to increases in the kinetic constants. 

Rhaman and coworkers [112,113] studied the adsorption of lipase on [MgAl] LDH and its biocatalytic activity for butyl oleate synthesis. They demonstrated that up to 277 and 531 mgg-1 of lipase were adsorbed on [MgAl-N03] and [MgAl-Dodecylsulfate] LDH, respectively, showing the highest adsorption capacity of the anionic clays compared to smectite or inorganic phosphate. Recently, we reported the adsorption isotherms of urease on [ZnRAl] LDH under various experimental conditions (pH, buffer) [117]. The kinetic study showed the fast adsorption process (less than 60 min) (Figure 15.3). 

Fig. 15.3 Kinetic study of urease adsorption on Zn2AI and Zn3AI LDH.

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

Gianfreda L, Rao MA, Violante A (1992) Adsorption, activity, and kinetic properties of urease on montmorillonite, aluminum hydroxides, and Al(OH)x-montmorillonite complexes. Soil Biol Biochem 24 51-58... 

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. 

Rates of reaction of urease with deuterated and especially tritiated urea were markedly reduced compared with the rate with the unlabeled ( H) substrate, but usually isotope effects are insignificant biochemically except in rigorous kinetic studies. 

For reactions in which one or more reactants or products is a gas, manometry (the measurement of pressure differences) can provide a convenient means for monitoring the course and kinetics of the reaction Thus, enzymes that can be assayed with this method include oxidases, urease, carbonic anhydrase, hydrogenase, and decarboxylases. For example, bacterial glutamate decarboxylase is readily assayed by utilizing a Warburg flask and measuring the volume of gas evolved at different times using a constant-pressure respirometer. ... 

BASAL RATE UPCONVERSION UREA KINETICS UREASE... 

Urea in kidney dialysate can be determined by immobilizing urease (via silylation or with glutaraldehyde as binder) on commercially available acid-base cellulose pads the process has to be modified slightly in order not to alter the dye contained in the pads [57]. The stopped-flow technique assures the required sensitivity for the enzymatic reaction, which takes 30-60 s. Synchronization of the peristaltic pumps PI and P2 in the valveless impulse-response flow injection manifold depicted in Fig. 5.19.B by means of a timer enables kinetic measurements [62]. Following a comprehensive study of the effect of hydrodynamic and (bio)chemical variables, the sensor was optimized for monitoring urea in real biological samples. A similar system was used for the determination of penicillin by penicillinase-catalysed hydrolysis. The enzyme was immobilized on acid-base cellulose strips via bovine serum albumin similarly as in enzyme electrodes [63], even though the above-described procedure would have been equally effective. 

Huang, T.C. and Chen, D.H., Kinetic studies on urea hydrolysis by immobilized urease in a batch squeezer and flow reactor, Biotechnol. Bioeng., (1992) 40,10,1203-09. Storey, K.B., Duncan, J.A., and Chakrabarti, A., Immobilization of amyloglucosidase using two forms of polyurethane polymer, Appl. Biochem. Biotechnol., (1990) 23, 3, 221-36. 

In another kinetics study, Huang and Chen immobilized jack bean urease in the form of a thin film on the surface of a reticulated polyurethane foam. The residual apparent activity of the urease after immobilization was about 50%. The good hydrodynamic properties and flexibility of the support were retained in solution after immobilization. Urea hydrolysis was examined in both a batch squeezer and circulated flow reactor. The results suggest potential for practical applications in various reactors. 

More recently, the kinetics of the urease-catalyzed decomposition of urea were investigated using a wide-range bipolar pulse instrument capable of both current and voltage pulse modes with either integrated or sampled data acqui-... 

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. 

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). 

An alternative scenario was put forward based on the crystal structures of urease inhibited by either phosphate, diamidophosphate, or borate (4,5, 28). It gets some support from the kinetic findings for fluoride inhibition of urease (29), as well as from recent model calculations (30, 31). Boric acid, known to be a competitive inhibitor of urease, can be considered a good substrate analogue, since it is isoelectronic with urea and has the same shape and dimension. Bacillus pasteurii could be crystallized in the presence of boric acid. The structure reveals that a molecule of B(OH)3 is symmetrically spanning the nickel ions, replacing Wj, W2,... 

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. 

Detection limits in EIA are ultimately determined by how low one can measure the label s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, /8-galactosi-dase, peroxidase, - and glucose oxidase. This is due to the high mo-... 

Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. 

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 ... 

A good example of the range of parameters available from flow calorimetric data can be found from the study of enzyme/substrate systems. The kinetic nature of enzyme systems has been previously described by Michaelis and Menten. In the treatment discussed here, the parameters sought are the enthalpy, rate constant, Michaelis constant and the enzyme activity. The following example describes a study on the well-known enzyme substrate system, urea/urease. 

The sensitivity of this method is directly related to the apparent molar enthalpy of reaction, so that very endo- or exothermic reactions will be most readily followed. Examples of the application of this method to the determination of enzyme kinetic parameters include dihydrofolate reductase, creatine phosphokinase, hexo-kinase, urease, trypsin, HIV-1 protease, heparinase, and pyruvate carboxylase. 

Enzymatic methods for the measurement of urea are based on preliminary hydrolysis of urea with urease (urea amidohydrolase, EC 3.5.1.5 main source jack bean meal) to generate ammonium ion, which is then quantified. This approach has been used in end-point, kinetic, conductimet-ric, and dry chemistry systems. ... 

Tsuji K, Soya Y, et al. Kinetic assay of serum and urine for urea with use of urease and leucine dehydrogenase. Clin Chem 1997 43 1932-6. 

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