The major
wound-inducible
monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms
geranyl pyrophosphate to both (-)-
alpha-pinene (40%) and (-)-
beta-pinene (60%). The
enzyme was purified to apparent homogeneity by
anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native
polyacrylamide gel electrophoresis at neutral pH and
polyacrylamide gel electrophoresis in the presence of
sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1%
Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured
enzyme produced a mixture of isomeric pinenes from
geranyl pyrophosphate identical to that generated by the native form. The
protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The
enzyme required Mn2+ (Km = 30 microM) for activity, exhibited a Km value of 6 microM for the substrate
geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 degrees C, and was inhibited by
pyrophosphate (I50 = 0.17 mM),
orthophosphate (I50 = 51 mM), and
alpha-pinene, as well as by the
histidine-directed
reagent diethylpyrocarbonate (I50 = 0.64 mM) and the
cysteine-directed
reagent p-hydroxymercuribenzoate (I50 = 1.9 microM). Although similar in many respects to constitutive
monoterpene cyclases of herbaceous species, this inducible cyclase, the first
enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent
metal ion cofactor.