Determination of the Number of Sucrose and Acceptor Binding Sites for Leuconostoc mesenteroides B-512FM Dextransucrase, and the Confirmation of the Two-Site Mechanism for Dextran Synthesis

In previous studies on dextransucrase using pulse and chase experiments with [14C]sucrose, Robyt et al. [Arch. Biochem. Biophys. 165 (1974) 634-640] proposed a two-site insertion mechanism to explain the data for the synthesis of dextran. To further establish the validity of the two-site mechanism, the number of sucrose binding sites at the active site have been determined by using equilibrium dialysis with 6-deoxysucrose, a strong competitive inhibitor for dextransucrase. A ligand binding plot gave a straight line that indicated there were two sucrose binding sites at the active site. A similar experiment was performed using the acceptor, maltose. The ligand binding plot for maltose also gave a straight line and indicated that there was one acceptor binding site at the active site. These results corroborate the proposed two-site mechanism for dextran synthesis. To further test the two-site mechanism, dextransucrase was partially inactivated to varying extents by reaction with diethylpyrocarbonate, which chemically modifies essential active-site histidines. The various partially inactivated enzymes were assayed for dextran synthesis and for the synthesis of maltose acceptor products. A plot of the log of the relative percentage of dextran synthesized and acceptor products synthesized against varying degrees of enzyme inactivation showed that the synthesis of dextran decreased to a greater extent than did the decrease of the synthesis of acceptor product. The proposed mechanism requires two sucrose sites for the synthesis of dextran and only one sucrose site for the synthesis of acceptor product. When one site is modified, the synthesis of dextran stops, but the synthesis of acceptor products can continue at the other site. Thus, the greater loss of dextran synthesis in comparison with the lesser loss of acceptor product synthesis by enzymes modified to varying degrees, gives further evidence for the two-site mechanism for dextran synthesis.