ENZYMIC EPIMERIZATION OF SEQUOYITOL TO D-PINITOL IN TRIFOLIUM INCARNATUM

The partial purification of an enzyme system from Trifolium incarnatum catalyzing the epimerization of sequoyitol to d‐pinitol and its characterization is described. The reaction proceeds via a keto intermediate, d‐5‐O‐methyl‐2,3,5/4,6‐pentahydroxycyclohexanone, and is catalyzed by two dehydrogenases or one protein with two dehydrogenase actions, of which the first one is NAD‐specific and oxidizes sequoyitol to the keto intermediate, whereas the other has the function to reduce the keto compound to d‐pinitol using NADPH as hydrogen donor. Both dehydrogenase actions as well as the overall epimerization reaction were shown to be reversible. Although the sequoyitol dehydrogenase activity can be easily destroyed by several means which leave the d‐pinitol dehydrogenase activity intact, no real separation of the two activities has been achieved. The protein or proteins responsible for the two activities have a molecular weight of about 34,000 as determined by the gel filtration method. The two activities do not only differ in their sensitivity towards inhibitors, their substrate and coenzyme specificities, but also in their stereospecificity with respect to the coenzymes. Whereas the NAD‐specific sequoyitol dehydrogenase activity is B‐specific, the NADP‐specific d‐pinitol dehydrogenase activity shows A‐specificity. On the basis of these findings, a possible mechanism for the epimerization is discussed. Copyright © 1969, Wiley Blackwell. All rights reserved