Purification and characterization of the nickel-containing multicomponent urease from Klebsiella aerogenes.

M.J. Todd,R.P. Hausinger

doi:10.1016/s0021-9258(18)45522-6

Abstract

Klebsiella aerogenes urease was purified 1,070-fold with a 25% yield by a simple procedure involving DEAE-Sepharose, phenyl-Sepharose, Mono Q, and Superose 6 chromatographies. The enzyme preparation was comprised of three polypeptides with estimated Mr = 72,000, 11,000, and 9,000 in a alpha 2 beta 4 gamma 4 quaternary structure. The three components remained associated during native gel electrophoresis, Mono Q chromatography, and Superose 6 chromatography despite the presence of thiols, glycols, detergents, and varied buffer conditions. The apparent compositional complexity of K. aerogenes urease contrasts with the simple well-characterized homohexameric structure for jack bean urease (Dixon, N. E., Hinds, J. A., Fihelly, A. K., Gazzola, C., Winzor, D. J., Blakeley, R. L., and Zerner, B. (1980) Can. J. Biochem. 58, 1323-1334); however, heteromeric subunit compositions were also observed for the enzymes from Proteus mirabilis, Sporosarcina ureae, and Selemonomas ruminantium. K. aerogenes urease exhibited a Km for urea of 2.8 +/- 0.6 mM and a Vmax of 2,800 +/- 200 mumol of urea min-1 mg-1 at 37 degrees C in 25 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid, 5.0 mM EDTA buffer, pH 7.75. The enzyme activity was stable in 1% sodium dodecyl sulfate, 5% Triton X-100, 1 M KCl, and over a pH range from 5 to 10.5, with maximum activity observed at pH 7.75. Two active site groups were defined by their pKa values of 6.55 and 8.85. The amino acid composition of K. aerogenes urease more closely resembled that for the enzyme from Brevibacter ammoniagenes (Nakano, H., Takenishi, S., and Watanabe, Y. (1984) Agric. Biol. Chem. 48, 1495-1502) than those for plant ureases. Atomic absorption analysis was used to establish the presence of 2.1 +/- 0.3 mol of nickel per mol of 72,000-dalton subunit in K. aerogenes urease.

Highlights

From the Department of Microbiology and Public Health and Department of Biochemistry, Michigan State University, East Lansing, Michigan48824-1101
Klebsiella aerogenesurease was purified 1,070-fold = 90,790) [5]. This planetnzyme contains two nickel ions per with a 25% yieldby a simpleprocedureinvolving subunit [6], and thme etal ions are apparentlycoordinated by DEAE-Sepharose, phenyl-Sepharose,Mono Q, and Su- oxygen and nitrogenligands [7]
The enzyme preparation appears tobe analogous in size, structure, and nickel content was comprised of three polypeptides with estimateMd

Summary

Introduction

The Selenom o w ruminantium urease is alsosmaller (native M , = 360,000), but like the plant enzymes it contains two nickel ions per subunit (Mr= 70,000) [11].Active site differences may exist in the microbial and plant enzymes as shown In addition,Kamel and Hamed[14] described v,, mM and a of 2,800 f200 pmol of urea min” mg” several properties of 150-fold-purified urease fromthe related a t 3 7“Cin 2 5 mM N-2-hydroxyethylpiperazine-N’-2- organism, Aerobacter aerogenes PRL-R3.

Results

Conclusion