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

Abstract

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.

Structural characteristics of the hydroxamic acid group. Crystal structure of formohydroxamic acid

“…the iron-containing enzyme ribonucleotide reductase (Kjoller Larsen, Sj6berg & Thelander, 1982), the nickel-containing enzyme urease (Dixon, Hinds, Fihelly, Gazzola, Winzor, Blakeley & Zerner, 1980), and zinc-or manganese-containing aminopeptidases Baker, Wilkes, Bayliss & Prescott, 1983). The inhibitory effect 0108-7681/88/050527-07503.00 of hydroxamic acids on ribonucleotide reductase has been shown to be due to the ability of the compounds to react with (reduce) the tyrosine free-radical group present in the subunit B2 of this enzyme (Kjoller Larsen et al, 1982).…”

The structure of jack bean urease

“…However, the subunit structure of this enzyme has been somewhat ambiguous. The relative molecular mass reported first for the native urease was 480000 [2] while recently the value of 590 000 was reported [3]. The values reported for the monomer range over 30000-97000 [3-61.…”

Purification and Characterization of Acid Urease fromLactobacillus reuteri

“…Recently, bacterial neutral ureases were found to consist of three kinds of subunit in a a 2 {3 4 y 4 structure, 11 • 12 l whereas jack bean urease is a hexamer of the unit protein. 13 l The approximate subunit ratio of the acid urease was I : 2: 1, which was determined from the integrated intensities of the gel scan profile, and calculation based on the molecular weight of 220,000 obtained on gel filtration chromatography and the molecular weights of the three polypeptides estimated by SDS-PAGE. The result suggests that the acid urease consists of 2 mol of the 68,000-dalton peptide, 4 mol of the 16, 100-dalton peptide and 2 mol of the 8,800-dalton peptide.…”