Influence of polymolecular events on inactivation behavior of xylose isomerase from Thermotoga neapolitana 5068

The inactivation behavior of the xylose isomerase from Thermotoga neapolita na (TN5068 XI) was examined for both the soluble and immobilized enzyme. Po lymolecular events were involved in the deactivation of the soluble enzyme. Inactivation was biphasic at 95 degrees C, pH 7.0 and 7.9, the second phas e was concentration-dependent. The enzyme was most stable at low enzyme con centrations, however, the second phase of inactivation was 3- to 30-fold sl ower than the initial phase. Both phases of inactivation were more rapid at pH 7.9, relative to 7.0. Differential scanning calorimetry of the TN5068 X I revealed two distinct thermal transitions at 99 degrees and 109 degrees C . The relative magnitude of the second transition was dramatically reduced at pH 7.9 relative to pH 7.0. Approximately 24% and 11% activity were recov erable after the first transition at pH 7.0 and 7.9, respectively. When the TN5068 XI was immobilized by covalent attachment to glass beads, inactivat ion was monophasic with a rate corresponding to the initial phase of inacti vation for the soluble enzyme. The immobilized enzyme inactivation rate cor responded closely to the rate of ammonia release, presumably from deamidati on of labile asparagine and/or glutamine residues. A second, slower inactiv ation phase suggests the presence of an unfolding intermediate, which was n ot observed for the immobilized enzyme. The concentration dependence of the second phase of inactivation suggests that polymolecular events were invol ved. Formation of a reversible polymolecular aggregate capable of protectin g the soluble enzyme from irreversible deactivation appears to be responsib le for the second phase of inactivation seen for the soluble enzyme. Whethe r this characteristic is common to other hyperthermophilic enzymes remains to be seen. (C) 1999 John Wiley & Sons, Inc.