Substrate binding sites of Leuconostoc dextransucrase evaluated by inhibition kinetics.

Graphical analysis of inhibition kinetics for dextransucrase from Leuconostoc mesenteroides was done with typical inhibitors, competitive and noncompetitive.Based on the plots of Yonetani-Theorell and Semenza-Balthazar, mutual competition between the pairs of inhibitors of identical kinetic type was observed, while combination of competitive and noncompetitive inhibitors gave no significant mutual interactions.By the procedure ofNitta et aL, binding sites for competitive and noncompetitive inhibitors were shown to be distant from each other.Moreover, two noncompetitive inhibitors competed with each other for a single binding site on the enzyme.Although biphasic reciprocal plots may suggest rather complicated binding of various inhibitors, the results obtained by the three graphical methods are fully explained when competitive and noncompetitive inhibitors for substrate sucrose bind to the so-called donor-and acceptor-sites of dextransucrase, respectively.Dextransucrase (EC 2.4.1.5)from Leuconostoc mesenteroides catalyzes a glucosyl transfer reaction from sucrose to acceptor dextran and produces a high molecular weight dextran as shown in equation (1).1)Sucrose + (D-Glucose)n > D-Fructose + (D-Glucose)n + 1 (1 )In our previous paper,2) we reported the purification of dextransucrase from L. mesenteroides strain NRRLB-1416, which contained only a small amount of endogenous dextran.