Effects of substitution of aspartate‐440 and tryptophan‐487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis

FEBS Letters

Mattick, John S.¹  Diefenbach, Russell J.¹  Candy, Judith M.¹  Duggleby, Ronald G.¹ 

[1] Department of Biochemistry, University of Queensland, Brisbane 4072, Australia

关键词: Pyruvate decarboxylase;    Site-directed mutagenesis;    Thiamin diphosphate binding;    Zymomonas mobilis;    ADH;    alcohol dehydrogenase;    PDC;    pyruvate decarboxylase;    ThDP;    thiamin diphosphate;   

DOI  :  10.1016/0014-5793(92)80411-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 μM for thiamin diphosphate and from 4.21 to 45 μM for Mg²⁺. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding, cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine of glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.

【 授权许可】

Unknown