Abstract
Abstract The purification and properties of geranyl pyrophosphate synthetase are reported. This enzyme is purified simultaneously with the enzyme for the synthesis of farnesyl pyrophosphate. It does not, however, have geranylgeranyl pyrophosphate synthetase activity. This enzyme, obtained as a single band of protein by starch gel electrophoresis, catalyzes the formation of both geranyl and farnesyl pyrophosphates from dimethylallyl pyrophosphate and 4-14C-isopentenyl pyrophosphate. These products have been identified by paper and gas-liquid chromatography. A pH range of 7.0 to 7.8 is optimum for the reaction. A metal ion is required and either Mg++ or Mn++ may be used, but Mn++ inhibits the reaction at concentrations greater than 4 x 10-3 m. Km values for isopentenyl and dimethylallyl pyrophosphates are 1.25 x 10-6 and 2.2 x 10-6 m, respectively. N-Ethylmaleimide and p-chloromercuribenzoate strongly inhibit the synthetase reaction, but iodoacetamide has only a slight inhibitory effect. The s20,w value for the synthetase is 4.7.
Highlights
The present paper reports the purification of the enzyme system for the formation of geranyl and farnesyl pyrophosphates
The principal compounds found on gasliquid chromatography of these terpenols were linalool and nerolidol. These results show that the single protein eluted from starch gel is capable of synthesizing both geranyl and farnesyl pyrophosphates from isopentenyl and dimethylallyl pyrophosphates
Radioactivity was coincident with authentic geranyl and farnesyl pyrophosphates detected with Rosenberg’s reagent. It was previously suggested by Benedict etal. [1] that separate enzymes exist for the biosynthesis of geranyl and farnesyl pyrophosphates in pig liver
Summary
In an earlier publication from this laboratory, Benedict et al [1] reported the partial purification of an enzyme, isolated from hog liver, which catalyzes the formation of farnesyl pyrophosphate from isopentenyl and geranyl pyrophosphates. The partially purified enzyme did not have activity for the isomerization of isopentenyl pyrophosphate [2, 3] or the formation of geranyl or geranylgeranyl pyrophosphates. An earlier publication [4] had reported that a single enzyme system isolated from Micrococcuslysodeikticus catalyzed each of the condensations involved in the formation of geranyl, farnesyl, and geranylgeranyl pyrophosphates. 3,3’-Dimethylacrylic acid was obtained from the Aldrich Chemical Company and 2-14C mevalonic acid was purchased as the N ,N’-dibenzylethylenediamine salt from New England Nuclear Corporation, Iodoacetamide was secured from K and K Laboratories, and alkaline phosphatase was supplied by Worthington. Linalool, farnesol, and nerolidol were purchased from Fritzsche Brothers; geranylgeraniol and geranyllinalool were a generous gift from Hoffmann-La Roche and Company, Basel, Switzerland
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