Enzyme Chemistry of C-P Compound Synthesis in Rumen Protozoa

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1. Some properties and chromatographic behavior of the enzyme which catalyzes formation of 3-phosphonopyruvate (PnPY) in rumen protozoa were studied in an attempt to reveal the role of rumen protozoa in the characteristic metabolism of ruminants.2. In view of the difficulty of detection and quantitation of free PnPY by HPLC, PnPY-2,4-dinitrophenylhydrazone was separated on a reversed phase column and was detected at 377nm. 0.1 nmol of PnPY could be quantitated.3. A preliminary research with log phase cells of Tetrahymena, which is a close relative of rumen protozoa, demonstrated that the enzyme localized in the soluble fraction of the cells and required Mg ion and heat stable cofactors. The pH- activity profile of the crude enzyme was bell-shaped and the activity was maximal at about pH8.4. Mixed protozoa were collected from the contents of sheep rumen, and the cells were fractionated according to the procedures for Tetrahymena. The PnPY-forming enzyme localized in a soluble fraction of the cells, but the activity was very low compared to that in Tetrahymena. On gel permeation chromatography, the enzyme activity in the soluble fraction increased and exceeded the activity in Tetrahymena. The activity was maximal at about pH7.2 and the molecular weight was estimated at about 70k dalton.5. Results of ion-exchange chromatography revealed that the enzymes of both rumen protozoa and Tetrahymena were basic proteins.6. Natural occurrence of PnPY has now been established, and all the results were consistent with the biosynthetic mechanism of PnPY through an intramolecular rearrangement of phosphoenolphruvate. However, the final proof for this mechanism of PnPY synthesis must await isolation of the new enzyme, phosphoenolphruvate phosphomutase.