A tyrosine residue essential for catalytic activity in aminopeptidase A.
A tyrosine residue essential for catalytic activity in aminopeptidase A.
复制标题
对氨肽酶 A 的催化活性至关重要的酪氨酸残基。
作者：
G. Vazeux;X. Iturrioz;P. Corvol;C. Llorens
氨肽酶A（EC 3.4.11.7;阿帕）是一种130 kDa的膜结合锌酶，其含有在锌金属蛋白酶家族中保守的共有序列HEXXH（残基385-389）。在这个基序中，组氨酸残基和谷氨酸残基被证明分别参与锌配位和催化活性。用N-乙酰咪唑处理阿帕会导致酶活性丧失，这是由竞争性氨肽酶抑制剂amastatin阻止的，表明阿帕活性位点存在重要的酪氨酸、赖氨酸或半胱氨酸残基。酪氨酸残基以前被认为与氨肽酶N的酶活性有关。此外，小鼠阿帕与其他单锌氨肽酶的序列比对表明存在保守的酪氨酸（阿帕中的Tyr-471）。Tyr-471在阿帕中的功能作用是通过定点突变用苯丙氨酸（Phe-471）或组氨酸（His-471）残基取代该残基来研究的。动力学研究表明，这两种突变体的Km值是类似的野生型酶，而kcat值下降了三个数量级，并对应于自由能的变化的限速步骤由4.0和4.2千卡/摩尔（0.96和1.00千焦/摩尔）的Phe-471和His-471突变体分别。突变没有修改的抑制效力的硫醇含抑制剂，强烈螯合的活性位点的锌离子，而假定的模拟反应中间体的过渡态的推定类似物减少。总之，这些结果强烈表明，Tyr-471羟基通过与含氧阴离子相互作用稳定过渡态复合物参与催化。
Aminopeptidase A (EC 3.4.11.7; APA) is a 130 kDa membrane-bound zinc enzyme that contains the consensus sequence HEXXH (residues 385-389) conserved among the zinc metalloprotease family. In this motif, both histidine residues and the glutamic residue were shown to be involved respectively in zinc co-ordination and catalytic activity. Treatment of APA with N-acetylimidazole results in a loss of enzymic activity; this is prevented by the competitive aminopeptidase inhibitor amastatin, suggesting the presence of an important tyrosine, lysine or cysteine residue at the active site of APA. A tyrosine residue was previously proposed to be involved in the enzymic activity of aminopeptidase N. Furthermore sequence alignment of mouse APA with other monozinc aminopeptidases indicates the presence of a conserved tyrosine (Tyr-471 in APA). The functional role of Tyr-471 in APA was investigated by replacing this residue with a phenylalanine (Phe-471) or a histidine (His-471) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of both mutants were similar to that of the wild-type enzyme, whereas kcat values were decreased by three orders of magnitude and corresponded to a variation in free energy of the rate-limiting step by 4.0 and 4.2 kcal/mol (0.96 and 1.00 kJ/mol) for the Phe-471 and His-471 mutants respectively. The mutation did not modify the inhibitory potency of a thiol-containing inhibitor that strongly chelates the active-site zinc ion, whereas that of a putative analogue of the transition state presumed to mimic the reaction intermediate was reduced. Taken together, these results strongly suggest that the Tyr-471 hydroxy group participates in catalysis by stabilizing the transition state complex through interaction with the oxyanion.