Pyridoxal phosphate binding to wild type, W330F, and C298S mutants of Escherichia coli apotryptophanase: unraveling the cold inactivation

FEBS Letters

Erez, Tali¹  Parola, Abraham H.¹  Phillips, Robert S.² 

[1] Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84 105, Israel;Department of Chemistry, University of Georgia, Athens, GA 30602, USA

关键词: Tryptophanase;    Pyridoxal phosphate;    Cold inactivation;    Site-directed mutagenesis;    Stopped-flow kinetics;    Protein conformation and quaternary structure;    Tnase;    tryptophanase;    WT-Tnase;    wild type Tnase;    PLP;    pyridoxal 5′-phosphate;    Tricine;    N-[2-hydroxy-1;    1-bis (hydroxymethyl)-ethyl]-glycine;   

DOI  :  10.1016/S0014-5793(98)00931-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown