Jack beans urease

Until the discovery in 1975 of nickel in jack bean urease (which, 50 years previously, had been the first enzyme to be isolated in crystalline form and was thought to be metal-free) no biological role for nickel was known. Ureases occur in a wide variety of bacteria and plants, catalyzing the hydrolysis of urea,... 

Laufberger had tried to obtain the protein from horse liver, but it did not crystallize, and as he described to me when I met him in Prague some years ago, in those days everyone wanted to have protein crystals as a criteria of purity. Although James Sumner had crystallized jack bean urease in 1926, his preparations were somewhat impure, and it was only in the mid-1930s, when John Northrop and Moses Kubnitz showed that there is a direct correlation between the enzymatic activities of crystalline pepsin, trypsin and chymotrypsin that the protein nature of enzymes was generally accepted. 

Effect of Inhibitors on Arrhenius Plots. The presence of inhibitors can lead to nonlinear Arrhenius plots as well as an increase in E. For example, jack bean urease exhibits a linear Arrhenius plot in the absence of an inhibitor, but a curved plot in the presence of 34 mM sodium sulfite . 

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. 

Jack bean urease is a trimer or hexamer of identical 91-kDa subunits while that of the bacterium Klebsiella has an (a(32y2)2 stoichiometry. Nevertheless, the enzymes are homologous and both contain the same binickel catalytic center (Fig. 16-25).435-4373 The three-dimensional structure of the Klebsiella enzyme revealed that the two nickel ions are bridged by a carbamyl group of a carbamylated lysine. Like ribulose bisphos-phate carboxylase (Fig. 13-10), urease also requires C02 for formation of the active enzyme.438 Formation of the metallocenter also requires four additional proteins, including a chaperonin and a nickel-binding protein.438 439... 

Physicochemical studies on jack bean urease during the past 10 years undoubtedly were stimulated by the 1960 paper of Creeth and Nichol... 

There are two reports that hydroxamic acid inhibition is reversible 59,90) and one that inhibition is irreversible (91). The inhibition appears to be competitive. The rather extensive screening of 36 hydroxamic acids was accomplished with sword bean urease (90), but Proteus urease (92) and jack bean urease (59) also have been found to be inhibited by these specific inhibitors. Using tritium-labeled caprylohydroxamic acid and sword bean urease, Kobashi et al. (94) have shown the formation of an inactive complex containing two moles of inhibitor per mole of enzyme. 

The enzyme urease catalyzes the hydrolysis of urea to form carbamate ion (equation 32). At pH 7.0 and 38 °C, the urease-catalyzed hydrolysis of urea is at least 1014 times as fast as the spontaneous hydrolysis of urea. Jack bean urease is a nickel(II) metalloenzyme502 with each of its six identical subunits containing one active site and two metal ions, and at least one of these nickel ions is implicated in the hydrolysis. It has been suggested503 that all substrates for urease (urea, N-hydroxyurea, 7V-methylurea, semicarbazide formamide and acetamide) are activated towards nucleophilic attack on carbon as a result of O-coordination to the active nickel(II) site as in (155). Nickel(II) ions have been found504 to promote the ethanolysis and hydrolysis of N-(2-pyridylmethyl)urea (Scheme 39) and this system is considered to be a useful model for the enzyme. 

Hasnain, S.S. and Piggott, B. (1983) An EXAFS study of Jack Bean urease, a nickel metal-loenzyme. Biochem. Biophys. Res. Commun., 112, 279-283. 

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. 

Prior to the crystallization of jack bean urease it was assumed by the biochemical community that enzymes had no ordered structure. In 1965 the first crystallographic evidence for the mechanism by which enzymes work when Phillips and his group solved the lysozyme structure [6], Details of the structure indicated how the enzyme could bind the oligosaccharides present in its target, bacterial cell wall peptidoglycans, and could respond to the binding event by changing its structure. 

Fig. 4.2 The first enzyme to be characterized jack bean urease [7]...

In subsequent steps the initial carbamate product is further hydrolyzed to bicarbonate. Jack bean urease is a hexamer containing two nickel ions per monomeric subunit. It is unusually difficult to remove the metal atoms from the protein, requiring low pH and resulting in irreversible inactivation of the enzyme. There are indications that one nickel ion is more tightly bound than the other and, therefore, that the two sites are inequivalent. 

L-lactate and oxalate were also tested with lactate-2-monoxygenase and oxalate decarboxylase and excellent results were obtained. CPG columns were employed in both instances. Good linearity was obtained between 0.005 -1 mM for L-lac-tate [3] and between 0.1-3 mM for oxalate [24]. Similarly, urea was measured with a precision better than 1 % in the linearity range 0.01-200 mM using Jack bean urease. The reaction of urea with ethanol to produce ethylcarbamate is of interest in fermentation monitoring. 

Since NH4 is stable at pH 7, the solution could be strongly buffered during this experiment to control the pH at the specimen surface. More recently GC mode was used to study the effects of adsorption at a metal surface and the applied potential on enzyme activity (30). Jack Bean urease adsorbs... 

Biological catalysts have been postulated since the early 1800 s. The term enzyme was coined in 1878 to describe the component in yeast involved in the fermentation of sugar into alcohol. The enzyme jack bean urease, which catalyzes hydrolysis of urea, was crystallized in 1926. Comparison between chemical and enzymatic catalysis demonstrates the specificity of enzymatic catalysis... 

Dixon NE, Gazzola C, Blakeley RL and Zerner B (1975) Jack bean urease a metalloenzyme A simple biological role for nickel J Am Chem Soc 97 4131-4133. 

Coordinated cyanamide does not add OH" to form the N-bound urea complex since it de-protonates 5.2) to ve the unreactive [Co(NCNH)(NH3)5p ion, but in acid it readily loses NH to form N-coordinated cyanate (equation 27). The analogous dimethylcyanamide complex cannot deprotonate and adds OH" to form urea (Scheme 21), but this does not proceed further to coordinated carbamic add and NHMCj in a process analogous to that suggested for the function of Ni in the metalloenzyme jack bean urease (which catalyzes the convemioo of urea to these products), nor does it lose NHMc2 to form the cyanato complex as suggested by Balahura and Jordan for the similar urea derivatives (equation 28). Alternatively, in acid solution the protonated urea rapidly isomerizes to the O-coordinated complex (r,/2 a 40 s, 25 °C Scheme 21). This isomer undergoes OH -catalyzed hydrolysis at the metal rather than at acyl carbon, so the function of the metalloenzyme has not been duplicated in this system. A similar property is found for 0-bound urea (equation 29). ... 

The nickel(II)-containing enzyme jack bean urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide [Eq. (3)] by a factor of lO " at pH 7.0 and 38 °C. As it is believed that coordination of urea to Ni(II) is fundamental to... 

Jack bean urease (E.C.3.5.1.5) Is a metalloenzyme. A simple biological role for nickel J. Amer. Chem. Soc.. 

It was not until 1926 that enzymes were shown to be proteins. In that year James B. Sumner (1887-1955) of Cornell University purified and crystallized the enzyme urease from jack beans. Urease is a protein that catalyzes the hydrolysis of urea ... 

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