Mechanism of the action of Leuconostoc mesenteroides B-512FMC dextransucrase: kinetics of the transfer of d-glucose to maltose and the effects of enzyme and substrate concentrations

The kinetics of the reaction of Leuconostoc mesenteroides B-512FMC dextransucrase with sucrose were studied. This enzyme catalyzes the synthesis of dextran from sucrose with a kcat of 641 s−1 and the transfer of d-glucose from sucrose to maltose with a kcat of 1070 s−1. The enzyme was also found to catalyze two new reactions in the absence of sucrose, using dextran as the substrate; d-glucose was transferred from the non-reducing ends of dextran chains to maltose with a relatively low kcat of 3.2 s−1; and d-glucose was hydrolyzed from the non-reducing ends of dextran chains with a very low kcat of 0.085 s−1. Ping-pong/bi-bi kinetics of these reactions are consistent with the formation of a glucosyl–enzyme covalent intermediate. It is shown that an increase in the concentrations of both maltose and sucrose in the d-glucose transfer reaction to maltose gives an exponential decrease in the amount of dextran and a concomitant increase in the amount of acceptor products. It is further shown that increasing the amount of dextransucrase gives a decrease in the amount of dextran and an increase in the amount of acceptor products, after the sucrose has been consumed. This anomaly occurs because the relatively high amounts of enzyme catalyze the transfer of d-glucose from the non-reducing ends of the dextran chains to maltose, giving a decrease in the amount of dextran and an increase in the amount of acceptor product. Further, the high amounts of enzyme catalyze the hydrolysis of the d-glucose residues from the ends of the dextran chains, giving a decrease in the amount of dextran. These reactions are not observed when lower amounts of enzyme are used, as the reactions are much slower than the synthesis of dextran and the usual acceptor transfer reactions of d-glucose from sucrose to acceptor.