Investigation of the Tetrahymena Pyriformis 2-Aminoethylphosphonic Acid Biosynthetic Pathway and the P-C Bond Forming Enzyme Phosphoenolpyruvate Mutase

The biosynthetic pathway leading to 2-aminoethylphosphonate in Tetrahymena pyriformis was determined. A cell-free homogenate of T. pyriformis converted phosphoenolpyruvate to AEP in 37% yield, phosphonopyruvate to AEP in a 11 % yield and phophonoacetaldehyde to AEP in an 83% yield. The Tetrahymena pyriformis enzyme, PEP mutase was purified. The PEP Mutase catalyzes the rearrangement of phosphoenolpyruvate to phosphonopyruvate and the equilibrium constant is >500:1 in favor of phosphoenolpyruvate. To distinguish between an intra-and intermolecular reaction pathway for this process an equimolar mixture of [P= ^18O, C(2)- ^18 O]thiophosphonopyruvate and (all - ^16Q)thiophosphonopyruvate was reacted with the PEP mutase and the resulting products were analyzed by ^31P-NMR. The absence of the cross over product [C(2)-18O]thiophosphonoenolpyruvate in the product mixture was interpreted as evidence for an intramolecular reaction pathway. To distinguish between a concerted and stepwise intramolecular reaction pathway the pure enantiomers of the chiral substrate [P= ^18Q]thiophosphonopyruvate were prepared and the stereochemical course of their conversion to chiral [P= ^18O]thiophosphoenolpyruvate was determined. Based on the observed conversion of (Sp) - [P= ^18O]thiophosphonopyruvate to (Sp)-[P= ^18O]thiophosphoenolpyruvate and (Rp) - [P = ^18O]thiophosphonopyruvate to (Rp)[P= ^18O]thiophosphoenolpyruvate it was concluded that the PEP phosphomutase reaction proceeds with retention of the phosphorus configuration and therefore by a stepwise mechanism. The similar reactivity of the oxo and thio substituted phosphonopyruvate substrates (i.e., nearly equal Vmax) was interpreted to suggest that addition to the phosphorus atom is not rate limiting among the reaction steps. Lastly, single turnover experiments failed to trap a pyruvate in the PEP mutase reaction