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.
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 beta-mercaptoethanol is approximately 95 000. Essentially the same subunit molecular weight (approximately 93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride - beta-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.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008433:Mathematics; D008623:Mercaptoethanol / Q000378:metabolism; D008970:Molecular Weight; D009532:Nickel / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation; D014510:Urease / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D006877:Hydroxamic Acids; C030685:cinnamoylhydroxamic acid; C006358:acetohydroxamic acid; D008623:Mercaptoethanol; D009532:Nickel; D014510:Urease