PYRIDOXAL-PHOSPHATE BINDING TO WILD-TYPE, W330F, AND C298S MUTANTS OFESCHERICHIA-COLI APOTRYPTOPHANASE - UNRAVELING THE COLD INACTIVATION

The mechanism of pyridoxal phosphate (PLP) binding to apotryptophanase was investigated using stopped-flow kinetics with wild type (WT), W33 0F and C298S mutants. Based on the dependence of the rate constants on PLP concentrations for the fast and slow phases detected, two mechani stic schemes were proposed. For the WT and C298S mutant, the slow proc ess is due to an isomerization of the aldimine complex after its forma tion, and not to the binding to an alternative conformation of the apo enzyme, which is the case proposed for the W330F mutant, It is suggest ed that during the cold inactivation process a conformational change p recedes the aldimine bond cleavage. For the W330F apotryptophanase, an other conformational change occurs subsequent to the aldimine bond cle avage. (C) 1998 Federation of European Biochemical Societies.