Properties of Kaurene Synthetase from Marah macrocarpus Endosperm: Evidence for the Participation of Separate but Interacting Enzymes
| Autor: | John D. Duncan, Charles A. West |
|---|---|
| Rok vydání: | 1981 |
| Předmět: |
chemistry.chemical_classification
Enzyme complex biology Geranylgeranyl pyrophosphate Physiology Stereochemistry Size-exclusion chromatography Articles Plant Science Pyrophosphate Ent-kaurene synthase Copalyl diphosphate synthase chemistry.chemical_compound Enzyme chemistry Biochemistry Genetics biology.protein Polyacrylamide gel electrophoresis |
| Zdroj: | Plant Physiology. 68:1128-1134 |
| ISSN: | 1532-2548 0032-0889 |
| DOI: | 10.1104/pp.68.5.1128 |
| Popis: | 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 [ 14 C]geranylgeranyl pyrophosphate and [ 3 H]copalyl pyrophosphate were incubated simultaneously with kaurene synthetase preparations demonstrated that copalyl pyrophosphate derived from [ 14 C]geranylgeranyl pyrophosphate is more readily converted to kaurene than is exogenously added [ 3 H]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. |
| Databáze: | OpenAIRE |
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