β-Amyrin is widely distributed in plant kingdom. This triterpene is biosynthesized by the polycyclization reaction of (3S)-2,3-oxidosqualene that proceeds with regio- and stereospecific fashion. The substrate is folded in a chair-chair-chair-boat-boat conformation inside the catalytic reaction cavity. Many genes harboring β-amyrin synthase have been reported, but very little is known of the enzyme properties of pure β-amyrin synthase and the active sites for the catalysis. In this symposium, we report the successful isolation and the kinetic parameters of β-amyrin cyclase from Euphorbia tirucalli (monofunctional OSC) and the functional analyses of the active site residue (Phe728). The optimal catalytic conditions and kinetic parameters were as follows: 30℃, pH 7.0, 0.05% (w/v) Triton X-100, K_M: 27.6 μM, k_<cat>: 42.0 min^<-1> , k_<cat>/K_M: 1.52 μM^<-1> min^<-1>, specific activity: 352 nmol/min/mg. There are only two examples describing the kinetic data of OSC, i.e., human and bovine lanosterol synthases, but no report has appeared describing those of β-amyrin synthase and other OSCs. Based on the amino acid alignment, F728 is supposed to be an active site residue. Mutagenesis experiments of F into A, I and M resulted in significant loss of the enzyme activity, but the Tyr mutant had a high activity at the same level as the wild type, suggesting that Phe728 stabilizes the cation intermediates through cation/π interaction. From the mutant of F728H, various enzymic products were isolated that were generated mainly from dammarenyl, lupenyl and oleanyl cations. These results indicate that F728 residue is located at the D-E ring formation site and that this residue has a crucial role for stabilizing these cations through cation/π interaction. There is no report regarding the comparison of the enzymatic activities between OSCs and the mutants. The in vivo relative enzyme activities between the mutants and the wild-type were determined as follows. The expressed amounts of the proteins were estimated by a Western blotting method and the product amounts were determined by GC analyses. The product amounts were divided by the expressed protein amounts, which enabled to successfully determine the relative enzyme activities between each of the site-directed mutants.
