IDENTIFICATION OF ZINC-BINDING LIGANDS IN THE CLASS-II FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE OF ESCHERICHIA-COLI
IDENTIFICATION OF ZINC-BINDING LIGANDS IN THE CLASS-II FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE OF ESCHERICHIA-COLI
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DOI：
10.1016/0014-5793(93)81317-s
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发表时间：
1993-02-22
期刊：
影响因子：
3.5
通讯作者：
MARSHALL, KE
中科院分区：
文献类型：
--
作者：
BERRY, A;MARSHALL, KE
建立了大肠杆菌的表达和诱变系统。通过修饰载体pKfda（Biochem.J.257（1989）529-534）已经产生了大肠杆菌II类果糖-1，6-二磷酸醛缩酶。获得大量的II类醛缩酶（在粗提物中约1g/l），其性质与先前报道的天然存在的酶（Biochem.J.169（1978）633-641）的性质一致。该酶每个酶二聚体含有2个锌离子。我们已经调查了酶的锌结合位点的性质，通过定点突变。His-108，His-111，Cys-112和His-142通过序列比对和与其他已知的含锌酶的比较被鉴定为可能的锌结合配体。这些残基的突变鉴定出His-108和His-111为直接负责锌的紧密结合的两个配体。其他两个残基的突变仅对每个单体结合的锌的量产生很小的影响，并导致比活性的相应变化。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。
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.