TY - JOUR
T1 - Cold-induced enzyme inactivation
T2 - How does cooling lead to pyridoxal phosphate-aldimine bond cleavage in tryptophanase?
AU - Erez, Tali
AU - Gdalevsky, Garik Ya
AU - Hariharan, Chithra
AU - Pines, Dina
AU - Pines, Ehud
AU - Phillips, Robert S.
AU - Cohen-Luria, Rivka
AU - Parola, Abraham H.
N1 - Funding Information:
We wish to express our gratitude and appreciation to Professor Yuri M. Torchinsky for introducing us to the study of tryptophanase. This work was partially supported by the James Franck Center for Laser-Matter Interaction and by the Office of Naval Research of the USA (Grant no. NOOO14-96-1-80) to A.H.P.
PY - 2002/2/11
Y1 - 2002/2/11
N2 - 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.
AB - 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.
KW - Protein quaternary structure
KW - Pyridoxal phosphate
KW - Single photon spectrofluorometry
KW - Time resolved fluorescence anisotropy
KW - Tryptophanase
KW - W330F mutant tryptophanase
UR - http://www.scopus.com/inward/record.url?scp=0037060470&partnerID=8YFLogxK
U2 - 10.1016/S0167-4838(01)00325-9
DO - 10.1016/S0167-4838(01)00325-9
M3 - Article
AN - SCOPUS:0037060470
SN - 0167-4838
VL - 1594
SP - 335
EP - 340
JO - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
JF - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
IS - 2
ER -