Cold-induced enzyme inactivation: how does cooling lead to pyridoxal phosphate–aldimine bond cleavage in tryptophanase?

• Published in: Biochimica et biophysica acta Vol. 1594; no. 2; pp. 335 - 340
• Main Authors: Erez, Tali, Gdalevsky, Garik Ya, Hariharan, Chithra, Pines, Dina, Pines, Ehud, Phillips, Robert S, Cohen-Luria, Rivka, Parola, Abraham H
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 11.02.2002
• Subjects: Anisotropy; Binding Sites; Cold Temperature; Enzyme Stability; Mutation; Protein Conformation; Protein quaternary structure; Pyridoxal phosphate; Pyridoxal Phosphate - chemistry; Rotation; Single photon spectrofluorometry; Spectrometry, Fluorescence; Time resolved fluorescence anisotropy; Tryptophanase; Tryptophanase - chemistry; Tryptophanase - genetics; W330F mutant tryptophanase; W330F mutant tryptophanase; Tnase, tryptophanase; Single photon spectrofluorometry; Tryptophanase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]-glycine; Pyridoxal phosphate; Time resolved fluorescence anisotropy; W330F Tnase, W330F mutant Tnase; PLP, pyridoxal 5′-phosphate; wt-Tnase, wild-type Tnase; Protein quaternary structure; SOPC, S-( o-nitrophenyl)- l-cysteine; CD, circular dichroism
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(01)00325-9

The phenomenon of cold scission or cold lability, which entails a widespread variety of oligomeric enzymes, is still enigmatic. The effect of cooling on the activity and the quaternary structure of the pyridoxal 5′-phosphate (PLP)-dependent enzyme, tryptophanase (Tnase), was studied utilizing single photon counting time-resolved spectrofluorometry. Upon cooling of holo-wild-type (wt) Tnase and its W330F mutant from 25°C to 2°C, a reduction in PLP fluorescence lifetime and rotational correlation time as well as inactivation and dissociation from tetramers to dimers were observed for both enzymes. Fluorescence anisotropy invariably decreased as a consequence of cooling, whether it was accompanied by a slight decrease in activity without significant dissociation, or by a substantial decrease in activity that was associated with either a partial or major dissociation. These results support the suggested conformational change that precedes the PLP–aldimine bond scission. It is proposed that cold inactivation is initiated by the weakening of hydrophobic interactions, leading to conformational changes which are the driving force for the aldimine bond cleavage.