Studies of the cryptic allylic pyrophosphate isomerase activity of trichodiene synthase using the anomalous substrate 6,7-dihydrofarnesyl pyrophosphate

Publisher Website
Google Scholar
Abstract
Two enantiomeric analogues of farnesyl pyrophosphate (1) were tested as inhibitors and anomalous substrates of trichodiene synthase, which catalyzes the cyclization of trans,trans-farnesyl pyrophosphate (1) to the sesquiterpene hydrocarbon trichodiene (2). The reaction has been shown to involve preliminary isomerization of 1 to the tertiary allylic isomer nerolidyl pyrophosphate (3) which is cyclized without detectable release of the intermediate from the active site of the cyclase. Both (7S)-trans-6,7-dihydrofarnesyl pyrophosphate (7a) and (7R)-trans-6,7-dihydrofarnesyl pyrophosphate (7b), prepared from (3R)- and (3S)-citronellol (9a and 9b), respectively, proved to be modest competitive inhibitors of trichodiene synthase. The values of Ki(7a), 395 nM, and Ki(7b), 220 nM, were 10-15 times the observed Km for 1 and half the Ki of inorganic pyrophosphate alone. Incubation of either 7a or 7b with trichodiene synthase resulted in formation of a mixture of products which by radio/gas-liquid chromatographic and GC/selected ion mass spectrometric analysis was shown to be composed of 80-85% isomeric trienes 19-21 and 15-20% allylic alcohols 12 and 18. Examination of the water-soluble products resulting from incubation of 7a also revealed the generation of 24% of the isomeric cis-6,7-dihydrofarnesyl pyrophosphate (26). The combined rate of formation of anomalous alcoholic and olefinic products was 10% the Vmax determined for the conversion of 1 to 2. The results can be explained by initial enzyme-catalyzed isomerization of dihydrofarnesyl pyrophosphate (7) to the corresponding tertiary allylic isomer dihydronerolidyl pyrophosphate (8). Since the latter intermediate is unable to cyclize due to the absence of the 6,7-double bond, ionization of 8 and quenching of the resulting ion pair by deprotonation, capture of water, or collapse to the isomeric primary pyrophosphate esters will generate the observed spectrum of anomalous products.

This publication has 0 references indexed in Scilit: