Jack bean urease (EC 3.5.1.5). III. The involvement of active-site nickel ion in inhibition by β-mercaptoethanol, phosphoramidate, and fluoride

1980 ◽  
Vol 58 (6) ◽  
pp. 481-488 ◽  
Author(s):  
Nicholas E. Dixon ◽  
Robert L. Blakeley ◽  
Burt Zerner
Keyword(s):  
Dissociation Constant ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Jack Bean Urease ◽  
Nickel Ion ◽  
Nickel Ions ◽  
Jack Bean ◽  
Complex Fluoride ◽  
Spectral Changes ◽  
Hydrolysis Of

Interaction of β-mercaptoethanol with urease produces large, rapid and fully reversible spectral changes in that part of the electronic absorption spectrum which is associated with the tightly bound nickel ions. The spectrophotometrically determined value of the dissociation constant of the β-mercaptoethanol–urease complex (0.95 ± 0.05 mM at pH 7.12 and 25 °C) is in agreement with the Ki (0.72 ± 0.26 mM) for β-mercaptoethanol acting as a competitive inhibitor in the hydrolysis of urea. This constitutes direct evidence that the nickel in jack bean urease is at the active site. Inhibition of urease by phosphoramidate is slowly achieved and slowly reversed, and upon reactivation of the isolated phosphoramidate–urease complex, phosphoramidate is regenerated in good yield. Spectrophotometric experiments indicate that phosphoramidate binds to nickel ion in urease. Competition with β-mercaptoethanol was used to determine a dissociation constant (1.23 ± 0.10 mM at pH 7.12 and 25 °C) for a fluoride–urease complex in which fluoride ion also coordinates with an active-site nickel ion. Kinetic evidence is presented which indicates that in the presence of urea, a ternary complex (fluoride–urea–urease) is formed.

Jack bean urease (EC 3.5.1.5). V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds

1980 ◽  
Vol 58 (12) ◽  
pp. 1335-1344 ◽  
Author(s):  
Nicholas E. Dixon ◽  
Peter W. Riddles ◽  
Carlo Gazzola ◽  
Robert L. Blakeley ◽  
Burt Zerner
Keyword(s):  
Enzymatic Hydrolysis ◽  
Mechanism Of Action ◽  
Active Site ◽  
Ph Dependence ◽  
Jack Bean Urease ◽  
Nickel Ion ◽  
Jack Bean ◽  
First Time ◽  
Hydrolysis Of ◽  
Hydrolysis Of Urea

Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 °C, kcat has the values 5870, 85, 0.55, and 0.075 s−1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato–enzyme intermediate.

Jack bean urease (EC 3.5.1.5). IV. The molecular size and the mechanism of inhibition by hydroxamic acids. Spectrophotometric titration of enzymes with reversible inhibitors

1980 ◽  
Vol 58 (12) ◽  
pp. 1323-1334 ◽  
Author(s):  
Nicholas E. Dixon ◽  
John A. Hinds ◽  
Ann K. Fihelly ◽  
Carlo Gazzola ◽  
Donald J. Winzor ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Active Site ◽  
Binding Sites ◽  
Spectrophotometric Titration ◽  
Hydroxamic Acids ◽  
Jack Bean Urease ◽  
Equivalent Weight ◽  
Guanidinium Chloride ◽  
Jack Bean ◽  
Equilibrium Ultracentrifugation

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 ± 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of β-mercaptoethanol is ~95 000. Essentially the same subunit molecular weight (~93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride – β-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein–inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.

Synthesis, cytotoxic and urease inhibitory activities of some novel isatin-derived bis-Schiff bases and their copper(ii) complexes

MedChemComm ◽  
2016 ◽  
Vol 7 (5) ◽  
pp. 914-923 ◽  
Author(s):  
Humayun Pervez ◽  
Maqbool Ahmad ◽  
Sumera Zaib ◽  
Muhammad Yaqub ◽  
Muhammad Moazzam Naseer ◽  
...  
Keyword(s):  
Schiff Bases ◽  
Active Compound ◽  
Active Site ◽  
Binding Mode ◽  
Jack Bean Urease ◽  
Jack Bean ◽  
Inhibitory Activities ◽  
Urease Inhibitory

The putative binding mode of the most active compound 3b in the active site of Jack bean urease.

scholarly journals The nickel ion environment in jack bean urease

1984 ◽  
Vol 220 (2) ◽  
pp. 591-595 ◽  
Author(s):  
L Alagna ◽  
S S Hasnain ◽  
B Piggott ◽  
D J Williams
Keyword(s):  
Fine Structure ◽  
Structure Study ◽  
Jack Bean Urease ◽  
Model Compounds ◽  
Deprotonated Form ◽  
Nickel Ion ◽  
Edge Structure ◽  
Jack Bean ◽  
X Ray ◽  
X Ray Absorption

Preliminary results of an extended X-ray absorption fine structure (e.x.a.f.s.) and X-ray absorption near edge structure study of jack bean urease have recently been reported [Hasnain & Piggott (1983) Biochem. Biophys. Res. Commun. 112, 279]. These results indicate that the environment of the nickel ion in the enzyme is similar to that in the model compounds Ni(L)2(L')1(ClO4)1 (where L is 1-n-propyl-2-alpha-hydroxybenzylbenzimidazole and L' is the deprotonated form) and Ni(HMB)3(Br)2 (where HMB is 2-hydroxymethylbenzimidazole), the closest similarity being with Ni(L)2-(L')1(ClO4)1. A detailed e.x.a.f.s. analysis has now been carried out and the crystal structures of the two model compounds solved. These results are reported here.

scholarly journals The interaction of yeast hexokinase with Procion Green H-4G

1981 ◽  
Vol 197 (1) ◽  
pp. 203-211 ◽  
Author(s):  
Y D Clonis ◽  
M J Goldfinch ◽  
C R Lowe
Keyword(s):  
Dissociation Constant ◽  
Active Site ◽  
Adenine Nucleotides ◽  
The Other ◽  
Crystallographic Structure ◽  
High Concentration ◽  
Apparent Dissociation Constant ◽  
Spectral Changes ◽  
Procion Yellow ◽  
Yeast Hexokinase

1. A number of reactive triazine dyes specifically and irreversibly inactive yeast hexokinase at pH 8.5 and 33 degrees C. Under these conditions, the enzyme is readily inactivated by 100 microM-Procion Green H-4G, Blue H-B, Turquoise H-7G and Turquoise H-A, is less readily inactivated by Procion Brown H-2G. Green HE-4BD, Red HE-3B and Yellow H-5G and is not inactivated at all by Procion Yellow H-A. 2. The inactivation of hexokinase by Procion Green H-4G is competitively inhibited by the adenine nucleotides ATP and ADP and the sugar substrates D-glucose, D-mannose and D-fructose but not by nonsubstrates such as D-arabinose and D-galactose. 3. Quantitatively inhibited hexokinase contains approx. 1 mol of dye per mol of monomer of mol.wt. 51000. The inhibition is irreversible and activity cannot be recovered on incubation with high concentration (20 mM) of ATP or D-glucose. 4. Mg2+ protects the enzyme against inactivation by Procion Green H-4G but enhances the rate of inactivation by all the other Procion dyes tested. In the presence of 10 mM-Mg2+ the apparent dissociation constant between enzyme and dye is reduced from 199.0 microM to 41.6 microM. Binding of the dye to hexokinase is accompanied by characteristic spectral changes in the range 560-700 nm. 5. Mg2+ promotes binding of yeast hexokinase to agarose-immobilized Procion Green H-4G but not to the other dyes tested. Elution could be effected by omission of Mg2+ from the column irrigants or by inclusion of MgATP or D-glucose, but not by D-galactose. These effects can be exploited to purify hexokinase from crude yeast extracts. 6. The specific active-site-directed binding of triazine dyes to yeast hexokinase is interpreted in terms of the crystallographic structure of the hexokinase monomer.

Synthesis, biological evaluation and docking studies of some novel isatin-3-hydrazonothiazolines

RSC Advances ◽  
2016 ◽  
Vol 6 (65) ◽  
pp. 60826-60844 ◽  
Author(s):  
Maqbool Ahmad ◽  
Humayun Pervez ◽  
Sumera Zaib ◽  
Muhammad Yaqub ◽  
Muhammad Moazzam Naseer ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Mode ◽  
Docking Studies ◽  
Biological Evaluation ◽  
Jack Bean Urease ◽  
Jack Bean

The putative binding mode of compound 6i in the active site of Jack bean urease.

Jack bean urease (EC 3.5.1.5). I. A simple dry ashing procedure for the microdetermination of trace metals in proteins. The nickel content of urease

1980 ◽  
Vol 58 (6) ◽  
pp. 469-473 ◽  
Author(s):  
Nicholas E. Dixon ◽  
Robert L. Blakeley ◽  
Burt Zerner
Keyword(s):  
Nickel Content ◽  
Spectrophotometric Analysis ◽  
Jack Bean Urease ◽  
Nickel Ions ◽  
Jack Bean ◽  
Determination Of Nickel ◽  
Dry Ashing ◽  
Soluble Species ◽  
Inexpensive Procedure

A simple and inexpensive procedure for determination of microgram quantities of metal ions in proteins is described and tested with nickel and iron. The method involves (a) dry ashing in an oxygen atmosphere at 450–460 °C in Pyrex vessels, (b) conversion of the metal oxides or other compounds to readily soluble species, and (c) spectrophotometric analysis. An improved procedure for the direct spectrophotometric determination of nickel using dimethylglyoxime is accurate to ± 2% or better with samples of 1–5 μg of nickel. These techniques were used to determine that the nickel content of freshly prepared jack bean urease is 2.00 ± 0.12 g-at./96 600 g protein. This corresponds to 2.0 nickel ions per subunit. This result was confirmed by atomic absorption analysis, which also showed that calcium, manganese, cobalt, and iron are not present in significant amounts in urease.

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