Purification and Characterization of the Monoterpene Cyclase Gamma-Terpinene Synthase From Thymus Vulgaris

Arch Biochem Biophys. 1991 May 1;286(2):511-7. doi: 10.1016/0003-9861(91)90073-r.

Abstract

The monoterpene cyclase, gamma-terpinene synthase, from Thymus vulgaris (thyme) leaves was purified to apparent homogeneity by isoelectric focusing and dye-ligand, anion-exchange, hydrophobic interaction, and gel permeation chromatography. The enzyme has a native molecular weight of 96,000 as determined by gel permeation chromatography, and exhibited a specific activity of 538 nmol/h.mg protein (turnover number of approximately 0.01/s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two apparently identical subunits of Mr approximately 55,000. The protein was very hydrophobic, and possessed a pI value of 4.85 as determined by isoelectric focusing. Maximum activity was observed at pH 6.8 in the presence of 20 mM Mg2+; 5 mM Mn2+ could support catalysis, albeit at a much lower rate. The Km value for the substrate, geranyl pyrophosphate, was 2.6 microM. Cyclase activity was inhibited by cysteine- and histidine-directed reagents. Purified gamma-terpinene synthase also possessed the ability to cyclize geranyl pyrophosphate to small amounts of alpha-thujene and to lesser quantities of myrcene, alpha-terpinene, limonene, linalool, terpinen-4-ol, and alpha-terpineol, all of which appear to be coproducts of the reaction sequence leading to gamma-terpinene. In general properties, the gamma-terpinene synthase from thyme leaves resembles other monoterpene cyclases as well as sesquiterpene and diterpene cyclases.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkyl and Aryl Transferases*
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Isoelectric Focusing
  • Kinetics
  • Magnesium / pharmacology
  • Magnoliopsida
  • Molecular Weight
  • Plants / enzymology*
  • Substrate Specificity
  • Transferases / isolation & purification*
  • Transferases / metabolism

Substances

  • Transferases
  • Alkyl and Aryl Transferases
  • gamma-terpinene synthetase
  • Magnesium