2,4,6-Trinitrobenzenesulphonic acid as a probe for lysine at the active site of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

Modification of Leuconostoc mesenteroides NRRL B-512F dextransucrase with 2,4,6-trinitrobenzenesulphonic acid (TNBS) at pH 5.2 and 30 degrees C resulted in the loss of enzyme activity. The kinetic profiles of inactivation showed that the reaction followed pseudo-first order reaction. Absorption spectra of TNBS modified enzyme gave characteristic maxima at 367 nm. The inactivation could not be reversed by dilution or dialysis. The substrate sucrose, provided protection to the enzyme against inactivation by TNBS, indicating that the essential residues are present at or near the active site. The stoichiometric results indicated that four mol of lysine are modified per mol of dextransucrase upon complete inactivation. However, more than 50% of the activity loss was accompanied by modification of 1 lysine residue. All these approaches suggested that one lysine residue present near or at the active site is essential for the enzymatic activity of dextransucrase.