Properties of Kaurene Synthetase from Marah macrocarpus Endosperm: Evidence for the Participation of Separate but Interacting Enzymes

Abstract

Ent-kaurene is synthesized from geranylgeranyl pyrophosphate in a two step sequence catalyzed by kaurene synthetase; the first step (A activity) involves the conversion of geranylgeranyl pyrophosphate into the intermediate ent-trans labda-8(17), 13-dien-15-yl pyrophosphate (copalyl pyrophosphate) which is further cyclized to ent-kaurene in the second step (B activity). The resolution of enzyme fractions which catalyze each step independent of the other has been accomplished for the first time by means of QAE Sephadex A-50 chromatography and polyacrylamide gel electrophoresis of kaurene synthetase preparations from endosperm tissue of immature seed of Marah macrocarpus. Molecular weights for the A and B enzymes were each estimated as approximately 82,000 by means of gel filtration chromatography and sedimentation velocity determinations. Experiments in which [¹⁴C]geranylgeranyl pyrophosphate and [³H]copalyl pyrophosphate were incubated simultaneously with kaurene synthetase preparations demonstrated that copalyl pyrophosphate derived from [¹⁴C]geranylgeranyl pyrophosphate is more readily converted to kaurene than is exogenously added [³H]copalyl pyrophosphate. This implies that copalyl pyrophosphate derived from the catalytic site of the A enzyme is preferentially channelled to the B enzyme catalytic site for conversion to ent-kaurene, rather than freely equilibrating with a pool of copalyl pyrophosphate in the medium. Experiments in which the rates of the overall AB and independent B activities of kaurene synthetase preparations were measured as a function of total protein concentration further suggest that overall AB activity is catalyzed by an AB enzyme complex which is in equilibrium with free A and B enzymes. A model is proposed for M. macrocarpus kaurene synthetase in which separate but interacting A and B enzymes must associate for the efficient production of ent-kaurene from geranylgeranyl pyrophosphate.