Todd M J, Hausinger R P
J Biol Chem. 1987 May 5;262(13):5963-7.
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.
产气克雷伯菌脲酶通过一个简单的程序进行纯化，该程序涉及DEAE-琼脂糖、苯基-琼脂糖、Mono Q和Superose 6色谱法，纯化倍数达1070倍，产率为25%。酶制剂由三种多肽组成，估计分子量分别为72,000、11,000和9,000，呈α2β4γ4四级结构。尽管存在硫醇、二醇、去污剂以及不同的缓冲条件，但在非变性凝胶电泳、Mono Q色谱法和Superose 6色谱法过程中，这三种组分仍保持结合状态。产气克雷伯菌脲酶明显的组成复杂性与刀豆脲酶简单且特征明确的同六聚体结构形成对比（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）；然而，奇异变形杆菌、脲芽孢八叠球菌和反刍月形单胞菌的脲酶也观察到了异源亚基组成。在25 mM N-2-羟乙基哌嗪-N'-2-乙磺酸、5.0 mM EDTA缓冲液（pH 7.75）中，37℃下，产气克雷伯菌脲酶对尿素的Km为2.8±0.6 mM，Vmax为2800±200 μmol尿素·min-1·mg-1。该酶活性在1%十二烷基硫酸钠、5% Triton X-100、1 M KCl中以及pH 5至10.5范围内稳定，在pH 7.75时活性最高。通过其pKa值6.55和8.85确定了两个活性位点基团。与植物脲酶相比，产气克雷伯菌脲酶的氨基酸组成更类似于产氨短杆菌的脲酶（Nakano, H., Takenishi, S., and Watanabe, Y. (1984) Agric. Biol. Chem. 48, 1495 - 1502）。原子吸收分析用于确定产气克雷伯菌脲酶中每摩尔72,000道尔顿亚基含有2.1±0.3摩尔镍。
