Bartholomew B A, Smith M J, Long M T, Darcy P J, Trudgill P W, Hopper D J
Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed, U.K.
Biochem J. 1995 Apr 15;307 ( Pt 2)(Pt 2):603-8. doi: 10.1042/bj3070603.
Tropine dehydrogenase was induced by growth of Pseudomonas AT3 on atropine, tropine or tropinone. It was NADP(+)-dependent and gave no activity with NAD+. The enzyme was very unstable but a rapid purification procedure using affinity chromatography that gave highly purified enzyme was developed. The enzyme gave a single band on isoelectric focusing with an isoelectric point at approximately pH 4. The native enzyme had an M(r) of 58,000 by gel filtration and 28,000 by SDS/PAGE and therefore consists of two subunits of equal size. The enzyme displayed a narrow range of specificity and was active with tropine and nortropine but not with pseudotropine, pseudonortropine, or a number of related compounds. The apparent Kms were 6.06 microM for tropine and 73.4 microM for nortropine with the specificity constant (Vmax/Km) for tropine 7.8 times that for pseudotropine. The apparent Km for NADP+ was 48 microM. The deuterium of [3-2H]tropine and [3-2H]pseudotropine was retained when these compounds were converted into 6-hydroxycyclohepta-1,4-dione, an intermediate in tropine catabolism, showing that the tropine dehydrogenase, although induced by growth on tropine, is not involved in the catabolic pathway for this compound. 6-Hydroxycyclohepta-1,4-dione was also implicated as an intermediate in the pathways for pseudotropine and tropinone catabolism.
托品脱氢酶由铜绿假单胞菌AT3在阿托品、托品或托品酮上生长诱导产生。它依赖于NADP(+)，对NAD+无活性。该酶非常不稳定，但开发了一种使用亲和层析的快速纯化程序，可得到高度纯化的酶。该酶在等电聚焦时呈现单一条带，等电点约为pH 4。通过凝胶过滤法测得天然酶的相对分子质量为58,000，通过SDS/PAGE测得为28,000，因此由两个大小相等的亚基组成。该酶表现出较窄的特异性范围，对托品和去甲托品有活性，但对假托品、假去甲托品或一些相关化合物无活性。托品的表观Km为6.06 microM，去甲托品的表观Km为73.4 microM，托品的特异性常数（Vmax/Km）是假托品的7.8倍。NADP+的表观Km为48 microM。当[3-2H]托品和[3-2H]假托品转化为托品分解代谢的中间体6-羟基环庚-1,4-二酮时，这些化合物中的氘得以保留，这表明托品脱氢酶虽然由在托品上生长诱导产生，但不参与该化合物的分解代谢途径。6-羟基环庚-1,4-二酮也被认为是假托品和托品酮分解代谢途径中的中间体。
