Identification of zinc‐binding ligands in the Class II fructose‐ 1,6‐bisphosphate aldolase of Escherichia coli

FEBS Letters

Berry, Alan¹  Marshall, Karen E.¹ 

[1] Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK

关键词: Aldolase;    Zinc binding;    Protein engineering;   

DOI  :  10.1016/0014-5793(93)81317-S

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

An expression and mutagenesis system for the E. coli Class II fructose-1,6-bisphosphate aldolase has been created by modification of the vector pKfda (Biochem. J. 257 (1989) 529-534). Large amounts of Class II aldolase (about 1 g/l in crude extracts), with properties consistent with those previously reported for the naturally occurring enzyme (Biochem. J. 169 (1978) 633-641) are obtained. The enzyme contains 2 zinc ions per enzyme dimer. We have investigated the nature of the zinc-binding site of the enzyme by site-directed mutagenesis. His-108, His-111, Cys-112 and His-142 were identified as possible zinc-binding ligands by sequence alignments and comparisons with other known zinc-containing enzymes. Mutation of these residues identified His-108 and His-111 as two of the ligands directly responsible for the tight binding of zinc. Mutation of the other two residues results in only a small effect on the amount of zinc bound per monomer and a corresponding change in specific activity. These residues are, therefore, unlikely to be directly involved in zinc binding, but may be indirectly involved in some manner in the zinc-binding environment.

【 授权许可】

Unknown