Jennings I G, Kemp B E, Cotton R G
Olive Miller Protein Chemistry Laboratory, Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Parkville, Victoria, Australia.
Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5734-8. doi: 10.1073/pnas.88.13.5734.
A monoclonal anti-idiotype antibody, NS7, previously shown to mimic the binding of the pterin cofactor of phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been used to localize the cofactor binding site within the phenylalanine hydroxylase catalytic domain to a 27-amino-acid sequence that is highly conserved among the three aromatic amino acid hydroxylases. The binding of NS7 to a synthetic peptide corresponding to the phenylalanine hydroxylase sequence from residue 263 to residue 289 was blocked by the competitive inhibitor of phenylalanine hydroxylase enzyme activity, 7,8-dihydro-6,7-dimethylpterin. In addition this peptide competed with native phenylalanine hydroxylase for binding to 6,7-dimethyl-5,6,7,8-tetrahydropterin conjugated to a polyglutamate carrier. Application of this simple and direct approach to other enzymes is likely to greatly facilitate the identification of ligand binding sites on enzymes, which will significantly contribute to the understanding of enzyme structure-function relationships.
一种单克隆抗独特型抗体NS7，先前已证明其可模拟苯丙氨酸羟化酶（苯丙氨酸4-单加氧酶，EC 1.14.16.1）的蝶呤辅因子的结合，现被用于将苯丙氨酸羟化酶催化结构域内的辅因子结合位点定位到一个27个氨基酸的序列上，该序列在三种芳香族氨基酸羟化酶中高度保守。苯丙氨酸羟化酶酶活性的竞争性抑制剂7,8-二氢-6,7-二甲基蝶呤可阻断NS7与对应于苯丙氨酸羟化酶第263位至第289位残基序列的合成肽的结合。此外，该肽与天然苯丙氨酸羟化酶竞争结合与聚谷氨酸载体偶联的6,7-二甲基-5,6,7,8-四氢蝶呤。将这种简单直接的方法应用于其他酶可能会极大地促进酶上配体结合位点的鉴定，这将对理解酶的结构-功能关系有显著贡献。
