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

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Abstract
Interaction of .beta.-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 .beta.-mercaptoethanol-urease complex (0.95 .+-. 0.05 mM at pH 7.12 and 25.degree. C) is in agreement with the Ki (0.72 .+-. 0.26 mM) for .beta.-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 .beta.-mercaptoethanol was used to determine a dissociation constant (1.23 .+-. 0.10 mM at pH 7.12 and 25.degree. 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.