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

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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.