MONOTERPENE BIOSYNTHESIS - ISOTOPE EFFECTS ASSOCIATED WITH BICYCLIC OLEFIN FORMATION CATALYZED BY PINENE SYNTHASES FROM SAGE (SALVIA-OFFICINALIS)

The three pinene synthases (cyclases) from common sage (Salvia officin alis) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins (+)-alpha-pinene and (+)-camphene (cyclase I), (-)-alpha-pine ne, (-)-beta-pinene, and (-)-camphene (cyclase II), and (+)-alpha-pine ne and (+)-beta-pinene (cyclase III), in addition to smaller amounts o f monocyclic and acyclic monoterpene olefins. [1-H-3,4-H-2(2)]- and [1 0-H-2(2)]-geranyl pyrophosphates were prepared and used in conjunction with 1-H-3- and 1-H-3,10-H-2(3)-labeled geranyl precursors to examine the isotope effects attending the C4- and C10-deprotonation steps in the enzymatic synthesis of the pinenes. The observation of isotopicall y sensitive branching within each set of stereochemically related bicy clic olefins confirmed that each product set was synthesized by the re spective pinene synthase by partitioning of common carbocationic inter mediates along different reaction channels at the active site. The cha nges in product distribution resulting from deuterium substitution at C4 and C10 of the substrate were used to determine kinetic isotope eff ects (KIEs) for the terminating deprotonations; these observed KIEs re present the lower limits of the intrinsic isotope effects. The intramo lecular isotope effects for the methyl-methylene elimination in beta-p inene formation by cyclases II and III were also evaluated with [10-H- 2(2)]geranyl pyrophosphate as substrate and by MS analysis of the olef in products. The intramolecular KIEs (k(H)/k(D) = 3.0 and 3.5) were si gnificantly higher than the observed KIEs determined from product rati os (k(Z)H/k(D) = 1.7 and 2.6) since the former involves considerably l ess masking of the intrinsic isotope effects. (C) 1994 Academic Press, Inc.