Jack bean urease (EC 3.5.1.5). Demonstration of a carbamoyl-transfer reaction and inhibition by hydroxamic acids

Blakeley, Robert L.; Hinds, John A.; Kunze, Hugo E.; Webb, Edwin C.; Zerner, Burt

doi:10.1021/bi00833a032

Cited by 137 publications

(66 citation statements)

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“…of 483000 was assumed. Although the present thiol-titration data might be taken to support the hexameric structure favoured by Blakeley et al (1969b), they do not of course preclude other urease structures. We shall subsequently refer to the 24 inessential and exposed thiol groups of urease as the class-I thiol groups, the six essential thiol groups as the class-II thiol groups, groups.…”

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confidence: 62%

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

Norris

Brocklehurst

1976

Biochemical Journal

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1. A convenient method ofpreparation ofjack-bean urease (EC 3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5±1.47kat/kg, Km = 3.32±0.05mM; kcat. = 2.15 x 104±0.05 x 104s-1 at pH7.0 and 38°C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558 suggests that the urease molecule (assumed to have mol.wt. 483000 and e280=2.84x105 litre-molhl cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-Il) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25'C (1= 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-Il thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 x 104M-l'S-1 and (b) pKa = 8.1, k = 8.05 x 102M-1 .-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60x 102M-I s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50 % class-Ia groups and 50 % class-lb groups. The ratio class Ia/class lb decreases as the pH is raised according to a pKa value > approx. 9.5, and at high pH the class-I thiol groups consist of at most 25 % class-Ia groups and at least 75 % class-lb groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be reactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.Urease (EC 3.5.1.5) can be extracted fromjack-bean (Canavalia ensiformis) meal and reproducibly purified to high specific activity by the method of Blakeley et al. (1969a). A chloroform/acetone-dried powder of jack-bean meal is extracted with 30% (v/v) acetone containing 1 % 2-mercaptoethanol at 39°C for 5min. The filtrate is left for 48h at 4°C to provide crystalline material that is then dissolved in buffer, dialysed and further purified by recycling upward-flow gel filtration on Sephadex G-200.We have reported a simple general method (covalent chromatography by thiol-disulphide interchange) for the purification ofthiol enzymes and its application in the immobilization of commercial samples of urease

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confidence: 62%

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

Norris

Brocklehurst

1976

Biochemical Journal

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“…This finding leads us to believe that the diabetogenic potential of ammonia may result, in part at least, by the intermediary of the catecholamines. However, in the ammonia intoxications caused by urease injections, one cannot eliminate with certainty any particular influence of urease or other metabolite arising from its action [5].…”

Section: Discussionmentioning

confidence: 99%

Diabetogenic effect and inhibition of insulin secretion induced in normal rats by ammonium infusions

Schlienger¹,

Imler²,

Stahl³

1975

Diabetologia

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Summary. In order to explain the abnormalities of glucose metabolism previously observed in patients with blood ammonia elevation, the effect of a transitory hyperammonemia on I. V. glucose tolerance was investigated in rats. An I. V. glucose tolerance test was performed in 3 groups of 15 rats 60 min after the beginning of a 95 rain infusion of either a 2 ml isotonic NaC1 solution (control group) or ammonium acetate solutions at low (0.50 gmol/kg/min. NI-I4+ ) or high doses (1.70 ~tmol/kg/min NH4+ ). The "high" NH4+ infusion produced an increase of blood ammonia to levels near 1000 ~tg/100 ml, a significant decrease in the K coefficient for glucose disappearance (2.53 x 10-24-0.20 compared to 4.92 x 10 -a 4-0.13 in control group) and a suppression of the radioimmunological plasma insulin (I. R. I.) response to glucose.With the "low" NH 4 -/-infusion the hyperammonemia was less pronounced (200-300 gg/100 ml), but the decrease in K(3.02 x 10 -2 4-0.15) and in the first phase of I.R.I. release remained significant. The decrease in glucose disappearance rate could be accounted for by the proportional decrease in insulin secretion. Thus glucose intolerance induced by ammonium acetate infusions may be due to a direct effect of NI"I 4 "1-on the pancreas. These abnormalities in glucose metabolism depend on the quantity of infused ammonium.Key words: Rat, ammonium infusion, blood ammonia, glucose metabolism, plasma immunoreactive insulin.In 1955 Bessman et al [4] suggested that the neurotoxicity of hyperammonemia in porto-systemic encephalopathy results from a disturbance in cerebral glucose utilization. The observation, in hepatic coma, of a decrease in cerebral oxygen consumption [12,22] and an increase in some intermediates of glucose metabolism [11,35] supports this hypothesis. However the influence of variations in ammonium metabolism on the regulation of carbohydrate metabolism is still debated [10,34]. Recently, we noted that I. V. glucose tolerance was worse in cirrhotic patients with hepatic coma than in those without porto-systemic encephalopathy [30]. One factor which could explain this observation, was the increase in arterial blood ammonia which was consistently observed during hepatic coma. Thus we noted in both normal and cirrhotic subjects, infused with ammonium chloride or acetate, that a transitory elevation in arterial blood ammonia invariably brought about a very significant diminution in I. V. glucose tolerance [31] and an inhibition of the insulin secretion induced by the glucose load [32]. However, in these studies, we were unable to show any correlation between these anomalies and the blood ammonia levels. The aim of * Presented at the 9th Meeting of the European Association for the Study of the Liver. Hemsedal, Norway, september [6][7] 1974 the present study was to compare in normal rats the action of a moderate or gross hyperammonemia on glucose tolerance and insulin secretion. Material and MethodsThree groups of 15 male Wistar rats, weighing 250-300 g and fasted for 20 hours, were used. Afte...

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“…1,24,[26][27][28][29][30] It is well known that the uncatalyzed reaction proceeds through an elimination pathway, and a cyanate intermediate has been identified in this case. 17,[31][32][33][34] The increase in pH arising from this reaction causes a broad range of deleterious effects.…”

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Catalyzed Decomposition of Urea. Molecular Dynamics Simulations of the Binding of Urea to Urease

Estiú

Merz

2006

Biochemistry

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We present the results of molecular dynamics simulations on the urea/urease system. The starting structure was prepared from the 2.0Å crystal structure of Benini et al. of DAP-inhibited urease (PDB code 3UBP), 1 and the trimeric structure (2479 residues) resulted in 180K atoms after solvation by water. The force field parameters were derived using the bonded model approach described by Hoops et al. 2 Three different systems were analyzed, each one modeling a different protonation pattern for the His320 and His219 residues. In each case, the three monomers of urease have been analyzed separately. The time averaged structures observed in the three monomers suggest that urease could follow two different competitive mechanisms. A "protein assisted proton transfer" mechanism points to Asp221 as crucial for catalysis. An "Asp mediated proton transfer" involves the transfer of a proton from the bridging OH to a NH 2 moiety of urea, assisted by Asp360 in the active site. The impact of the simulation results on our understanding of urease catalysis are discussed in detail.The reliable prediction of the mechanism involved in the selectivity and efficiency of enzyme catalyzed reactions continues to challenge both experimental and computational researchers. 3-9 Mutagenic and biochemical procedures, as well as a variety of computational methodologies have been applied with the goal of understanding the enzymatic effect on reactivity, and several working hypotheses have been proposed. Among them, there is general agreement of the role played by the reduction of the free energy of activation, 3 but the origins of the free energy lowering, which involves non-covalent and covalent effects, is the subject of ongoing debate. 10 Non-covalent factors involve transition state electrostatic stabilization, 11 ground state destabilization and desolvation, 12 reduction of reorganization energy by binding in near attack conformations, 13 entropy trapping, 14 as well as several other related effects. Covalent and non-covalent effects are not exclusive, and rate enhancement can come from both effects, as well as from others. Covalent factors are dominant, whenever present. 15 They usually occur in metalloenzymes, where the metal centers covalently bind the substrate, stabilizing the transition state complex and leading to highly proficient catalysts. 10, 16 This is the case of the metalloenzyme urease, whose proficiency is presently a matter of extensive discussion, as values of 10 17 and 10 32 have been determined by experimental and theoretical means. 17, 18Urea amidohydrolase (urease) is a nickel-containing enzyme that catalyses the hydrolysis of urea to produce ammonia and carbon dioxide in the last step of nitrogen mineralization. 1, 19-25 The mechanism of the catalyzed reaction continues to be of interest, and there is still no agreement on whether the reaction involves a carbamic acid intermediate, a cyanate * To whom correspondence should be addressed. .edu † Present address: Department of Chemistry, Quantum Theory Project, ...

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Jack bean urease (EC 3.5.1.5). Demonstration of a carbamoyl-transfer reaction and inhibition by hydroxamic acids

Cited by 137 publications

References 22 publications

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

Diabetogenic effect and inhibition of insulin secretion induced in normal rats by ammonium infusions

Catalyzed Decomposition of Urea. Molecular Dynamics Simulations of the Binding of Urea to Urease

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