Ž . Biochimica et Biophysica Acta 1384 1998 365–372 Cold inactivation and dissociation into dimers of Escherichia coli tryptophanase and its W330F mutant form Tali Erez a , Garik Ya. Gdalevsky b , Yuri M. Torchinsky b , Robert S. Phillips c , Abraham H. Parola a, ) a Department of Chemistry, Ben-Gurion UniÕersity of the NegeÕ, P.O. Box 653, Beer-SheÕa 84105, Israel b Institutes for Applied Research, Ben-Gurion UniÕersity of the NegeÕ, P.O. Box 653, Beer-SheÕa 84105, Israel c Departments of Chemistry and of Biochemistry and Molecular Biology, UniÕersity of Georgia, Athens, GA 30602, USA Received 16 December 1997; accepted 18 February 1998 Abstract The kinetics and mechanism of reversible cold inactivation of the tetrameric enzyme tryptophanase have been studied. Cold inactivation is shown to occur slowly in the presence of K q ions and much faster in their absence. The W330F mutant tryptophanase undergoes rapid cold inactivation even in the presence of K q ions. In all cases the inactivation is accompanied by a decrease of the coenzyme 420-nm CD and absorption peaks and a shift of the latter peak to shorter wavelengths. The spectral changes and the NaBH test indicate that cooling of tryptophanase leads to breaking of the 4 internal aldimine bond and release of the coenzyme. HPLC analysis showed that the ensuing apoenzyme dissociates into dimers. The dissociation depends on the nature and concentration of anions in the buffer solution. It readily occurs at low protein concentrations in the presence of salting-in anions Cl y , NO y and I y , whereas salting-out anions, especially HPO 2y , 3 4 hinder the dissociation. K q ions do not influence the dissociation of the apoenzyme, but partially protect holotryptophanase from cold inactivation. Thus, the two processes, cold inactivation of tryptophanase and dissociation of its apoform into dimers exhibit different dependencies on K q ions and anions. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Tryptophanase; Pyridoxal phosphate; Cold lability; Mutant enzyme; Protein dissociation 1. Introduction Ž Tryptophanase tryptophan indole-lyase, EC . 4.1.99.1 is a widely distributed bacterial PLP-depen- dent enzyme consisting of four identical 52 kDa Abbreviations: Tnase, tryptophanase; WT-Tnase, wild type Tnase; W330F-Tnase, W330F-mutant Tnase; PLP, pyridoxal 5 X - w phosphate; CD, circular dichroism; Tricine, N- 2-hydroxy-1,1- Ž . x bis hydroxymethyl -ethyl -glycine ) Corresponding author. Fax: q972-7-6472943; E-mail: aparola@bgumail.bgu.ac.il monomers; dissociation into monomers occurs only wx at denaturing conditions or at pH above 8.7 1 . Each monomer contains one molecule of PLP, which forms an aldimine bond with a Lys residue; due to this aldimine Tnase exhibits pH-dependent absorption and w x CD spectra with maxima at 420 and 337 nm 1–3 . Ž q q Tnase requires certain monovalent cations K , NH , 4 q . w x Tl for its activity and for tight PLP binding 4–6 . There are conflicting reports in the literature about wx the behavior of Tnase in cold. Morino and Snell 7 found that E. coli apo-Tnase, but not the holoen- zyme, dissociates into dimers in dilute solutions at 0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. Ž . PII S0167-4838 98 00031-4