Bruce N C, Wilmot C J, Jordan K N, Stephens L D, Lowe C R
Institute of Biotechnology, University of Cambridge, U.K.
Biochem J. 1991 Mar 15;274 ( Pt 3)(Pt 3):875-80. doi: 10.1042/bj2740875.
The NADP(+)-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M10. A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P. putida M10. Morphine dehydrogenase was found to be a monomer of Mr 32,000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine. The pH optimum of morphine dehydrogenase was 9.5, and at pH 6.5 in the presence of NADPH the enzyme catalyses the reduction of codeinone to codeine. The Km values for morphine and codeine were 0.46 mM and 0.044 mM respectively. The enzyme was inhibited by thiol-blocking reagents and the metal-complexing reagents 1,10-phenanthroline and 2,2'-dipyridyl, suggesting that a metal centre may be necessary for activity of the enzyme.
在恶臭假单胞菌M10以葡萄糖为培养基生长的细胞中检测到了依赖烟酰胺腺嘌呤二核苷酸磷酸（NADP(+)）的吗啡脱氢酶，该酶催化吗啡氧化为吗啡酮。开发了一种快速可靠的纯化方法，该方法包括在固定化染料上进行两个连续的亲和色谱步骤，用于从恶臭假单胞菌M10中纯化该酶，纯化倍数达1216倍，达到电泳纯。发现吗啡脱氢酶是一种分子量为32,000的单体，对底物具有高度特异性，仅氧化吗啡和可待因的C-6羟基。吗啡脱氢酶的最适pH为9.5，在pH 6.5且存在烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的情况下，该酶催化可待因酮还原为可待因。吗啡和可待因的米氏常数（Km）分别为0.46 mM和0.044 mM。该酶受到硫醇阻断剂以及金属络合剂1,10-菲啰啉和2,2'-联吡啶的抑制，这表明金属中心可能是该酶活性所必需的。
