title: Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.
abstract: (4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
authors.forename: J I
authors.initials: JI
authors.lastname: Rajaonarivony
authors.forename: J
authors.initials: J
authors.lastname: Gershenzon
authors.forename: J
authors.initials: J
authors.lastname: Miyazaki
authors.forename: R
authors.initials: R
authors.lastname: Croteau
mesh_terms: D001665:Binding Sites; D003545:Cysteine / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D006639:Histidine / Q000494:pharmacology; D019753:Intramolecular Lyases; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D012395:Rose Bengal / Q000494:pharmacology
chemical_list: D012395:Rose Bengal; D006639:Histidine; D007535:Isomerases; D019753:Intramolecular Lyases; C040265:pinene cyclase I; C044526:geranyl-diphosphate cyclase; D003545:Cysteine; D004047:Diethyl Pyrocarbonate