Identification of catalytic cysteine, histidine, and lysine residues in Escherichia coli homoserine transsuccinylase.
Identification of catalytic cysteine, histidine, and lysine residues in Escherichia coli homoserine transsuccinylase.
复制标题
DOI：
10.1021/bi0620252
复制
发表时间：
2007-02
期刊：
影响因子：
2.9
通讯作者：
Katharine Ziegler;Schroeder M Noble;E. Mutumanje;B. Bishop;D. Huddler;T. Born
中科院分区：
文献类型：
--
作者：
Katharine Ziegler;Schroeder M Noble;E. Mutumanje;B. Bishop;D. Huddler;T. Born
高丝氨酸转移琥珀酸酶在几种细菌中催化高丝氨酸的琥珀酸化，这是这些生物中蛋氨酸生物合成的第一个独特的步骤。据报道，这种来自大肠杆菌的酶是一种二聚体，采用乒乓催化机制，将琥珀酸从琥珀酰辅酶A转移到亲核酶，然后转移到高丝氨酸形成O-琥珀酰高丝氨酸。用定点突变和稳态动力学方法鉴定了参与催化作用的三种氨基酸。半胱氨酸-142突变为丝氨酸或丙氨酸，消除了所有可测量的活性，表明这种氨基酸起到了催化亲核试剂的作用。半胱氨酸亲核体经常被组氨酸残基去质子化，组氨酸-235被鉴定为家族成员中唯一绝对保守的组氨酸残基。这一残基被突变为丙氨酸和天冬酰胺，两个突变体都没有观察到活性。赖氨酸-47此前已被确定为必需残基。将这种氨基酸突变为精氨酸，催化活性降低了90%以上，而突变为丙氨酸，产生了一种酶，其活性仅为野生型的1%。K47R突变体的pH-速率谱表明，该氨基酸参与了前半反应。本文提供的数据首次详细描述了高丝氨酸转移琥珀酸酶活性部位，并为该酶的其他机制表征提供了一个框架。
Homoserine transsuccinylase catalyzes the succinylation of homoserine in several bacterial species, the first unique step in methionine biosynthesis in these organisms. The enzyme from Escherichia coli is reported to be a dimer and uses a ping-pong catalytic mechanism involving transfer of succinate from succinyl-CoA to an enzyme nucleophile, followed by transfer to homoserine to form O-succinylhomoserine. Site-directed mutagenesis and steady-state kinetics were used to identify three amino acids that participate in catalysis. Mutation of cysteine-142 to serine or alanine eliminated all measurable activity, suggesting this amino acid acts as the catalytic nucleophile. Cysteine nucleophiles are often deprotonated by histidine residues, and histidine-235 was identified as the sole absolutely conserved histidine residue among family members. This residue was mutated to both alanine and asparagine, and no activity was observed with either mutant. Lysine-47 had been previously identified as an essential residue. Mutation of this amino acid to arginine reduced catalytic activity by greater than 90%, while mutation to alanine yielded an enzyme with <1% of wild-type activity. A pH-rate profile of the K47R mutant demonstrated that this amino acid participates in the first half reaction. The data presented here provide the first detailed description of the homoserine transsuccinylase active site and provide a framework for additional mechanistic characterization of this enzyme.