Urease kinetic studies

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.

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. 

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. 

Huang, T.-C., and D.-H. Chen. 1992. Kinetic Studies on Urea Hydrolysis by Immobilized Urease in a Batch Squeezer and Flow Reactor. Biotechnology and Bioengineering 40 (10) 1203-1209. 

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. 

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. 

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). 

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. 

Kulys et al. (1986b) studied urea determination by difference measurement between two antimony electrodes covered with exchangable membranes (Fig. 68). Urease was attached in the pores of a macroporous membrane (thickness, 10 pm, pore diameter, 0.1 pm) by glutaraldehyde. This layer was covered with a monoacetylcellulose membrane. The membrane for the auxiliary electrode was prepared analogously, but using BSA instead of urease. The assay of urea was carried out with a differential amplifier which simultaneously differentiated the time course of the potential difference between enzyme and auxiliary electrode (kinetic method). Thus, a response time of only 20 s was possible. 

Enzymes that hydrolyze proteins and other compounds composed of amino acids were among the first biological catalysts to be discovered, and they have continued to be prominent in studies of enzyme structure, kinetics, activation, and mechanism of action. The crystallization of the enzyme urease by Sumner was followed by the crystallization of various proteolytic enzymes in the laboratory of Northrop. These studies established that catalytic activity is associated with what appear to be pure proteins all well-defined enzymes isolated subsequently have also proved to be proteins, although many contain additional components. The study of enzymatic reactions involving proteins as substrate, therefore, gives insight into the chemical nature of enzymes as well as the mechanisms by which they act. 

If one of the reactants or products in a reaction is either hydroxide ions, OH (aq) or hydrogen ions, H (aq) (oxonium ions H30 (aq)), then there will be a change in pH (Chapter 8). This can be followed with a pH probe and meter. One interesting example of this is the study of the kinetics of reactions involving the enzyme urease. Since one of the products of enzyme action is ammonia, the reaction can be followed by monitoring the increased alkalinity of the solution. Note also that this type of reaction could also be followed by the sample removal/titration method mentioned below. 

Because most soil enzyme studies deal with crude soil suspensions, or at best partially-purified soil extracts, care must be exercised in assigning an activity to the action of a particular enzyme. This note of caution is perhaps justified by considering the conversion in soils of urea to NH4 and CO2. Urea is an important nitrogenous fertilizer and is a major constituent of the urine of grazing animals, and studies of urea hydrolysis have dominated the soil enzyme literature. It is widely assumed that urea hydrolysis is catalysed by urease (urea amidohydrolase, EC 3.5.1.5). Studies have compared the kinetics and inhibition of urea hydrolysis by soils and by purified ureases, mainly from jack bean, Canavalia ensiformis, Jack bean urease catalyses urea hydrolysis by a pathway in which carbamate is an intermediate. 

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