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

[8-H-3]-, [8,8,9,9-H-2(4)]-, and [1-H-3,8,9-H-2(6)] geranyl diphosphat es (1-t, 1-d4, and 1-d6,t) were synthesized and used as substrates for several monoterpene cyclases to determine the regiospecificity and is otope effects attending the terminating proton transfers in the enzyme -catalyzed biosynthesis of (R)- and (S)-limonene. Degradation of enant iomeric [H-3]limonenes produced by cyclization Of 1-t with the (+)- an d (-)-pinene cyclases (synthases) from Salvia officinalis demonstrated that the eliminations occur at both the cis- (55-65%) and trans-methy l (45-35%) groups. In contrast, the terminating eliminations in the fo rmation of (+)- and (-)-limonene catalyzed by limonene cyclasses from Citrus sinensis and Perilla frutescens, respectively, were shown by de gradation to occur exclusively (greater-than-or-equal-to 97-98%) at th e cis terminal methyl group. The intramolecular isotope effects for th e 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 determinati ons of [H-2]-limonene derived from enzymatic cyclizations of 1-d4. Sim ilar experiments with (-)-limonene cyclase from Mentha spicata resulte d in k(H)/k(D) = 4.0 +/- 0.4. Incubations of 1-d6,t with pinene and bo rnyl PP cyclases from S. officinalis exhibited significant remote isot ope effects (k(H)/k(D) = 1.16-1.27) on the total rate of monoterpene f ormation which suggest that the initial cyclization step of the enzyme -bound linalyl diphosphate intermediate is an important component of t he overall rate of the enzymatic reactions. The isotope effects on the partitioning of the alpha-terpinyl carbocation intermediate between b icyclization and elimination to limonene were determined from the effe cts of deuterium substitution on the product ratios derived from enzym atic cyclization of 1-d6,t. The small size of these product isotope ef fects (k(H)/k(D)) = 1.2-1.7) is attributed to a conformational inversi on of the alpha-terpinyl ion to a half-chair conformer prior to proton elimination to limonene, thereby rendering the bicyclizations relativ ely immune to the intrinsic deuterium isotope effect. The regiospecifi c proton transfers from the cis terminal methyl group effected by the limonene cyclases from Citrus and Perilla are attributed to the minimi zation of charge separation in the transition state.