Properties of Kaurene Synthetase from Marah macrocarpus

Abstract

The kaurene synthetase from immature seeds of Marah macrocarpus (Greene) Greene was partially purified from cell-free homogenates of endosperm by a combination of QAE-Sephadex A-25 chromatography and hydroxyapatite chromatography and freed of contaminating phosphatase activity. The two catalytic activities associated with kaurene synthetase, the cyclization of geranylgeranyl-pyrophosphate to copalyl-pyrophosphate (activity A) and the cyclization of copalyl-pyrophosphate to ent-kaurene (activity B), were not even partially resolved from one another during these procedures. Both activities had identical elution profiles from a calibrated Sepharose 4B column corresponding to a molecular weight less than that of ovalbumin (45,000).The A and B activities had pH optima of 7.3 and 6.9, respectively. Both activities required millimolar concentrations of the following divalent cations in the order: Mg(2+) > Mn(2+) > Co(2+). Activities A and B were both sensitive to inhibition by Hg(2+), Cu(2+), p-hydroxymercuribenzoate, and N-ethylmaleimide, but activity B was much more sensitive than activity A. The average value of Km' (apparent Km in the absence of substrate inhibition) for geranylgeranyl-pyrophosphate was 1.6 mum. Values of 0.5 and 0.6 mum were obtained for Km' and Km, respectively, for copalyl-pyrophosphate. The Vm' values for the two activities were similar: 12 and 9 pmol/minute.mug protein for activities A and B, respectively.N,N-Dimethylaminoethyl-2,2-diphenylpentanoate (SKF-525A) and N,N-dimethylaminoethyl-2,2-diphenylphentyl ether (SKF-3301A), tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D), tributyl-2,4-dichlorobenzylammonium chloride (Phosfon S), 2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (Amo-1618), 2-(N,N-dimethyl-N-heptylammonium bromide)-p-methan-1-ol (Q-58), and 2-(N,N-dimethyl-N-octylammonium bromide)-p-methan-1-ol (Q-64), at concentrations from 1 to 5 mum, were effective inhibitors of kaurene synthetase activity A. Acetylcholine chloride and 2-chloroethyl-trimethylammonium chloride were effective inhibitors of activity A only at concentrations of 5 mm or greater. Abscisic acid, indole-3-acetate, gibberellin A(1), gibberellin A(3), a mixture of gibberellins A(4) and A(7), gibberellin A(13), and N,N-dimethylaminosuccinamic acid (B995) were not inhibitory at any of the levels tested. None of these compounds was an effective inhibitor of activity B at concentrations less than 0.5 mm.