PMID:8436219
| Citation |
Berry, A and Marshall, KE (1993) Identification of zinc-binding ligands in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli. FEBS Lett. 318:11-6 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Cysteine/chemistry; Escherichia coli/enzymology; Fructose-Bisphosphate Aldolase/chemistry; Fructose-Bisphosphate Aldolase/metabolism; Histidine/chemistry; Ligands; Molecular Sequence Data; Plasmids; Sequence Homology, Amino Acid; Zinc/metabolism |
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