REGIOSPECIFICITY AND ISOTOPE EFFECTS ASSOCIATED WITH THE METHYL METHYLENE ELIMINATIONS IN THE ENZYME-CATALYZED BIOSYNTHESIS OF (R)-LIMONENE AND (S)-LIMONENE

[8-H-3]-, [8,8,9,9-H-2(4)]-, and [1-H-3,8,9-H-2(6)] geranyl diphosphates (1-t, 1-d4, and 1-d6,t) were synthesized and used as substrates for several monoterpene cyclases to determine the regiospecificity and isotope effects attending the terminating proton transfers in the enzyme-catalyzed biosynthesis of (R)- and (S)-limonene. Degradation of enantiomeric [H-3]limonenes produced by cyclization Of 1-t with the (+)- and (-)-pinene cyclases (synthases) from Salvia officinalis demonstrated that the eliminations occur at both the cis- (55-65%) and trans-methyl (45-35%) groups. In contrast, the terminating eliminations in the formation of (+)- and (-)-limonene catalyzed by limonene cyclasses from Citrus sinensis and Perilla frutescens, respectively, were shown by degradation to occur exclusively (greater-than-or-equal-to 97-98%) at the cis terminal methyl group. The intramolecular isotope effects for the methyl-methylene elimination in limonene biosynthesis catalyzed by (+)- and (-)-pinene cyclases from S. officinalis were found to be k(H)/k(D) = 2.3 +/- 0.2 and 5.9 +/- 0.5, respectively, by GC/MS determinations of [H-2]-limonene derived from enzymatic cyclizations of 1-d4. Similar experiments with (-)-limonene cyclase from Mentha spicata resulted in k(H)/k(D) = 4.0 +/- 0.4. Incubations of 1-d6,t with pinene and bornyl PP cyclases from S. officinalis exhibited significant remote isotope effects (k(H)/k(D) = 1.16-1.27) on the total rate of monoterpene formation which suggest that the initial cyclization step of the enzyme-bound linalyl diphosphate intermediate is an important component of the overall rate of the enzymatic reactions. The isotope effects on the partitioning of the alpha-terpinyl carbocation intermediate between bicyclization and elimination to limonene were determined from the effects of deuterium substitution on the product ratios derived from enzymatic cyclization of 1-d6,t. The small size of these product isotope effects (k(H)/k(D)) = 1.2-1.7) is attributed to a conformational inversion of the alpha-terpinyl ion to a half-chair conformer prior to proton elimination to limonene, thereby rendering the bicyclizations relatively immune to the intrinsic deuterium isotope effect. The regiospecific proton transfers from the cis terminal methyl group effected by the limonene cyclases from Citrus and Perilla are attributed to the minimization of charge separation in the transition state.