Abstract
Potato tuber UDP‐glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP‐glucose to MgPP~i~ forming glucose 1‐phosphate and MgUTP, according to an ordered bi‐bi mechanism in which UDP‐glucose and MgPP~i~ bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5′‐diphosphate, a reactive PP~i~ analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg^2+^ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPP~i~, and glucose 1‐phosphate, but enhanced by UDP‐glucose. The enhancement was prevented by co‐addition of P~i~, the competitive inhibitor with respect to PP~i~. The complete inactivation corresponded to the incorporation of 0.9–1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP‐glucose, labels were almost exclusively incorporated into Lys‐329. Thus, this residue may be located near the bound MgPP~i~ and its modification is promoted, probably through conformational changes, by the binding of UDP‐glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys‐329 and Lys‐263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPP~i~ and in the UDP‐glucose‐induced conformational changes, respectively.