Berry A, Marshall K E
Department of Biochemistry, University of Cambridge, UK.
FEBS Lett. 1993 Feb 22;318(1):11-6. doi: 10.1016/0014-5793(93)81317-s.
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.
通过对载体pKfda（《生物化学杂志》257卷（1989年）529 - 534页）进行改造，构建了一种用于大肠杆菌II类果糖 - 1,6 - 二磷酸醛缩酶的表达和诱变系统。可获得大量的II类醛缩酶（粗提物中约1 g/l），其性质与先前报道的天然存在的酶的性质一致（《生物化学杂志》169卷（1978年）633 - 641页）。该酶每个酶二聚体含有2个锌离子。我们通过定点诱变研究了该酶锌结合位点的性质。通过序列比对以及与其他已知含锌酶的比较，确定His - 108、His - 111、Cys - 112和His - 142为可能的锌结合配体。这些残基的突变确定His - 108和His - 111是直接负责锌紧密结合的两个配体。另外两个残基的突变仅对每个单体结合的锌量有较小影响，并导致比活性相应变化。因此，这些残基不太可能直接参与锌结合，但可能以某种方式间接参与锌结合环境。
