TROPINE DEHYDROGENASE - PURIFICATION, SOME PROPERTIES AND AN EVALUATION OF ITS ROLE IN THE BACTERIAL METABOLISM OF TROPINE

Tropine dehydrogenase was induced by growth of Pseudomonas AT3 on atro pine, tropine or tropinone. It was NADP(+)-dependent and gave no activ ity with NAD(+). The enzyme was very unstable but a rapid purification procedure using affinity chromatography that gave highly purified enz yme was developed. The enzyme gave a single band on isoelectric focusi ng 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 the refore 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 K(m)s were 6.06 mu M for tropine and 73.4 mu M for nortropine with the specificity constant (V-max/K-m) for tropine 7.8 times that for pseudotropine. The apparent K-m for NADP(+) was 48 mu M. The deuterium of [3-H-2]tropine and [3-H-2]pseudotropine was ret ained when these compounds were converted into 6-hydroxycyclohepta-1,4 -dione, an intermediate in tropine catabolism, showing that the tropin e dehydrogenase, although induced by growth on tropine, is not involve d in the catabolic pathway for this compound. 6-Hydroxycyclohepta-1,4- dione was also implicated as an intermediate in the pathways for pseud otropine and tropinone catabolism.