羽扇豆碱羟化酶，一种来自降解生物碱的假单胞菌属的喹诺细胞色素c
Lupanine hydroxylase, a quinocytochrome c from an alkaloid-degrading Pseudomonas sp.
作者信息
Hopper D J, Rogozinski J, Toczko M
机构信息
Department of Biochemistry, University College of Wales, Aberystwyth, Dyfed, U.K.
出版信息
Biochem J. 1991 Oct 1;279 ( Pt 1)(Pt 1):105-9. doi: 10.1042/bj2790105.
Lupanine 17-hydroxylase, the first enzyme in the pathway for bacterial degradation of the alkaloid, lupanine, was purified from a Pseudomonas sp. The enzyme acts by initial dehydrogenation of the substrate, and cytochrome c was used as electron acceptor in assays. It had an Mr of 66,000 by ultracentrifuge studies and 74,000 by gel filtration. The visible absorption spectrum was that of a cytochrome c, and a stoicheiometry of one haem group per molecule of enzyme was calculated. SDS/PAGE gave a single band of Mr 72,000 containing the haem group. The enzyme also contained pyrroloquinoline quinone (PQQ), which could be removed by isoelectric focusing. The apoenzyme was reconstituted to full activity with addition of PQQ, and a stoicheiometry of one molecule of PQQ per molecule of enzyme was calculated. Steady-state kinetics gave values of 3.6 microM for the Km for lupanine, 21.3 microM for the Km for cytochrome c and 217 s-1 for the Kcat.
羽扇豆碱17-羟化酶是生物碱羽扇豆碱细菌降解途径中的第一种酶，它是从一种假单胞菌中纯化得到的。该酶通过首先使底物脱氢起作用，在测定中细胞色素c用作电子受体。通过超速离心研究其Mr为66,000，通过凝胶过滤为74,000。可见吸收光谱是细胞色素c的光谱，计算得出每分子酶含有一个血红素基团的化学计量。SDS/PAGE给出了一条Mr为72,000的单带，其中含有血红素基团。该酶还含有吡咯并喹啉醌（PQQ），可通过等电聚焦去除。通过添加PQQ将脱辅基酶重构为具有完全活性的酶，并计算得出每分子酶含有一分子PQQ的化学计量。稳态动力学得出羽扇豆碱的Km值为3.6 microM，细胞色素c的Km值为21.3 microM，Kcat为217 s-1。
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