Biosynthesis of monoterpenes: inhibition of (+)-pinene and (-)-pinene cyclases by thia and aza analogs of the 4R- and 4S-alpha-terpinyl carbocation.
(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4R)- and the (4S)-thia and -aza analogs of the alpha-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.
McGeady
P
P
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Pyun
H J
HJ
Coates
R M
RM
Croteau
R
R
D001372:Aza Compounds / Q000138:chemical synthesis / Q000494:pharmacology; D007202:Indicators and Reagents; D019753:Intramolecular Lyases; D007535:Isomerases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013729:Terpenes / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*
D001372:Aza Compounds; D007202:Indicators and Reagents; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D013729:Terpenes; C077643:alpha-terpinyl pyrophosphate; C015234:geranyl pyrophosphate; D007535:Isomerases; C040245:pinene cyclase II; D019753:Intramolecular Lyases; C040265:pinene cyclase I