Levansucrase of Bacillus subtilis. Characterization of a stabilized fructosyl-enzyme complex and identification of an aspartly residue as the binding site of the fructosyl group.
A covalently linked fructosyl-enzyme 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 shown to be 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. However stability measurements of the fructosyl-enzyme bond indicated a marked lability at pH values above 8.5. The apparent rate constant of the hydrolytic reaction of this bond evaluated under the standard state of molar concentration of hydroxide ion was of the same order of magnitude as the apparent rate constant of the hydrolytic reaction of the transient fructosyl-enzyme postulated from the kinetic analysis of levansucrase. Furthermore, 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 we have isolated in a high state of purity. 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 cyanogen bromide released only one fructosylated peptide. The apparent molecular weight of this peptide was estimated to be lower than 10000.
D001224:Aspartic Acid / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D001665:Binding Sites; D005632:Fructose / Q000378:metabolism*; D006602:Hexosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding
D005632:Fructose; D001224:Aspartic Acid; D006602:Hexosyltransferases