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Pyridoxal phosphate binding to wild type, W330F, and C298S mutants of Escherichia coli apotryptophanase: unraveling the cold inactivation
Author(s) -
Erez Tali,
Phillips Robert S.,
Parola Abraham H.
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00931-4
Subject(s) - escherichia coli , mutant , chemistry , biochemistry , pyridoxal 5 phosphate , pyridoxal phosphate , phosphate , wild type , pyridoxal , enzyme , cofactor , gene
The mechanism of pyridoxal phosphate (PLP) binding to apotryptophanase was investigated using stopped‐flow kinetics with wild type (WT), W330F and C298S mutants. Based on the dependence of the rate constants on PLP concentrations for the fast and slow phases detected, two mechanistic schemes were proposed. For the WT and C298S mutant, the slow process is due to an isomerization of the aldimine complex after its formation, and not to the binding to an alternative conformation of the apoenzyme, which is the case proposed for the W330F mutant. It is suggested that during the cold inactivation process a conformational change precedes the aldimine bond cleavage. For the W330F apotryptophanase, another conformational change occurs subsequent to the aldimine bond cleavage.