COLD INACTIVATION AND DISSOCIATION INTO DIMERS OF ESCHERICHIA-COLI TRYPTOPHANASE AND ITS W330F MUTANT FORM

The kinetics and mechanism of reversible cold inactivation of the tetr americ enzyme tryptophanase have been studied. Cold inactivation is sh own to occur slowly in the presence of K+ ions and much faster in thei r absence. The W330F mutant tryptophanase undergoes rapid cold inactiv ation even in the presence of K+ ions. In all cases the inactivation i s accompanied by a decrease of the coenzyme 420-nm CD and absorption p eaks and a shift of the latter peak to shorter wavelengths, The spectr al changes and the NaBH4 test indicate that cooling of tryptophanase l eads to breaking of the internal aldimine bond and release of the coen zyme. HPLC analysis showed that the ensuing apoenzyme dissociates into dimers, The dissociation depends on the nature and concentration of a nions in the buffer solution. It readily occurs at low protein concent rations in the presence of salting-in anions Cl-, NO3- and I-, whereas salting-out anions, especially HPO42-, hinder the dissociation. K+ io ns do not influence the dissociation of the apoenzyme, but partially p rotect holotryptophanase from cold inactivation. Thus, the two process es, cold inactivation of tryptophanase and dissociation of its apoform into dimers exhibit different dependencies on K+ ions and anions. (C) 1998 Elsevier Science B.V. All rights reserved.