Effects of Magnesium, Pyrophosphate and Phosphonates on Pyrophosphorolytic Reaction of UDP-Glucose Pyrophosphorylase

UDP-glucose pyrophosphorylase (UGPase) carries a freely reversible reaction, using glucose-1-P and UTP to produce UDP-glucose (UDPG) and pyrophosphate (PP_i), with UDPG being essential for glycosylation reactions in all organisms including, e.g., synthesis of sucrose, cellulose and glycoproteins. In the present study, we found that free magnesium (Mg²⁺) had profound effects on the reverse reaction of purified barley UGPase, and was absolutely required for its activity, with an apparent K_m of 0.13 mM. More detailed analyses with varied concentrations of MgPP_i allowed us to conclude that it is the MgPP_i complex which serves as true substrate for UGPase in its reverse reaction, with an apparent K_m of 0.06 mM. Free PP_i was an inhibitor in this reaction. Given the key role of PPi in the UGPase reaction, we have also tested possible effects of phosphonates, which are analogs of PPi and phosphate (P_i). Clodronate and etidronate (PP_i analogs) had little or no effect on UGPase activity, whereas fosetyl-Al (P_i analog), a known fungicide, acted as effective near-competitive inhibitor versus PP_i, with K_i of 0.15 mM. The data are discussed with respect to the role of magnesium in the UGPase reaction and elucidating the use of inhibitors in studies on cellular function of UGPase and related enzymes.