LEVANSUCRASE OF BACILLUS-SUBTILIS - CHARACTERIZATION OF A STABILIZED FRUCTOSYL-ENZYME COMPLEX AND IDENTIFICATION OF AN ASPARTYL RESIDUE AS BINDING-SITE OF FRUCTOSYL GROUP

A covalently linked fructosyl-enzyme [levansucrase, EC 2.4.1.10, of B. subtilis] complex was isolated from a reaction mixture of enzyme and sucrose submitted to the quenching effect of a large decrease of the pH. The fructosyl-enzyme bond was stable under acidic and neutral conditions in the presence of high concentration of urea and of sodium dodecyl sulfate. This intermediate did not transfer at a measurable rate its fructosyl group to the usual fructosyl acceptors of the enzyme reaction under the usual conditions of enzyme activity. Stability measurements of the fructosyl-enzyme bond indicated a marked lability at pH values > 8.5. The apparent rate constant of the hydrolytic reaction of this bond evaluated under the standard state of molar concentration of OH- was of similar magnitude as the apparent rate constant of the hydrolytic reaction of the transient fructosyl-enzyme postulated from the kinetic analysis of levansucrase. Nucleophilic agents like imidazole enhanced the hydrolytic reaction of the fructosyl-enzyme bond. Identification of the fructosyl binding site on the enzyme was accomplished by proteolytic hydrolysis of the trapped complex. Peptic digestion followed by pronase digestion released a fructosyl-aspartate compound that was isolated in a highly pure state. The lability of the fructosyl-aspartate bond under mild alkaline conditions suggested that the fructosyl was linked through an ester bond involving the .beta.-carboxyl of the aspartate residue. Treatment of the trapped complex with CNBr released only 1 fructosylated peptide. The apparent MW of this peptide was < 10,000.