Stereochemistry, isotope effects, and inhibition of terpene biosynthesis: I. Stereospecificity and mechanism of monoterpene cyclases. II. Inhibition by heteroatom analogs of carbocation intermediates

Abstract

Stereospecificity and mechanism of monoterpene cyclases have been investigated by use of deuterium and tritium labeled substrates. The stereochemistry of the terminating deprotonation in limonene biosynthesis was elucidated by radiochemical degradation of (\sp3H) carvoximes. Limonene cyclases extracted from Citrus sinensis and Perilla frutescens effected regiospecific deprotonation, but pinene cyclases I and II from Salvia officinalis deprotonate non-regiospecifically. Deuterium isotope effects on the monoterpene product distribution from incubations of (1-\sp3H;8,9-\sp2H\sb6) GPP with the pinene cyclases are too small to explain by isotopically sensitive branching between limonene and the bicyclic monoterpenes.(3R,4R)-($-$)- And (3S,4S)-(+)-(4-\sp2H\sb1)linalool were synthesized to elucidate the stereospecificity of the terminating deprotonations in $\alpha$-pinene biosynthesis. The deuterium content of $\alpha$-pinene derived from the deuterated LPPs shows that the deprotonation in (+)-$\alpha$-pinene biosynthesis occurs stereospecifically by removal of the C4-H\sb{\rm R} hydrogen of (3R)-LPP, but that in ($-$)-$\alpha$-pinene biosynthesis removal of the enantiomeric C4-H\sb{\rm S} hydrogen occurs with only 83% stereoselectivity. This non-stereospecific deprotonation suggests that a two-base mechanism may be involved in the competitive formation of $\alpha$- and $\beta$-pinene.Aza and thia analogs of presumed $\alpha$-terpinyl and the bisabolyl carbocation intermediates were synthesized via degradation of enantiomeric limonenes for studies of terpene biosynthesis. The enantiomeric aza and thia analogs of the $\alpha$-terpinyl cation and the aza analog of the bisabolyl cation exhibited synergistic inhibition with PPi in kinetic evaluations of their effects on the pinene cyclases and trichodiene synthase, respectively. However, comparison of the inhibition data for each enantiomeric pair showed only small differences in the binding affinities. The additional isoprenyl unit on the aza analog of the bisabolyl cation increases the binding affinity to trichodiene synthase judging from a 8-10 fold decrease in K\sb{\rm i}.3-Aza-GGPP was found to inhibit GGPP synthase selectively in the presence of FPP synthase. Comparison of the inhibitory effects of (E,E,E)-, (Z,E,E)-, and 3-aza-GGPP supports the notion of a carbocation intermediate in the chain elongation process. However, 3-aza-GGPP was not an effective inhibitor for dehydrodolichyl PP synthase and farnesyl:protein transferase. Although the analog was found to inhibit geranylgeranyl:protein transferase, its binding affinity was about 10 times weaker than that of GGPP. In preliminary assays, ($\pm$)-3-aza-2-farnesyl-FPP inhibited squalene synthase with an I\sb{50} of $\sim$20-25 $\mu$M. (Abstract shortened with permission of author.)U of I OnlyETDs are only available to UIUC Users without author permissio