Levansucrase of Bacillus subtilis: kinetic and thermodynamic aspects of transfructosylation processes.

Simple kinetic considerations derived from the ping-pong mechanism previously proposed for levansucrase of Bacillus subtillis allowed us to predict an easily operated method to approach the kinetic studies of exchange and hydrolytic activities of this enzyme. The experimental kinetic pattern obtained from the study of both activities is in close agreement with those predicted by theoretical approach. The combination of kinetic results enabled us to determine with a good accuracy the values of the apparent rate constant of the step of fructosylation of the enzyme from the sucrose-enzyme Michaelis complex and the apparent rate constants of the steps of defructosylation of the fructosyl enzyme to water or to glucose. The standard free energy reaction coordinate diagram for the transfructosylation process from sucrose to water was constructed. We found that the high energy of the glycosidic linkage of sucrose is preserved in the fructosyl-enzyme intermediate. The temperature dependence studies of the rate constants of fructosylation and defructosylation of the enzyme show that the entropy of activation for the two steps of defructosylation of the fructosyl enzyme are nearly the same. However the enthalpy of activation for the transfructosylation step to water is greater than that to glucose. We attempted to explain this discrepancy. Furthermore comparison of mechanism and efficiency of enzymatic and acid catalysis of sucrose hydrolysis was developed.