Histidine 450 plays a critical role in catalysis and, with Ca2+, contributes to the substrate specificity of aminopeptidase A
Histidine 450 plays a critical role in catalysis and, with Ca2+, contributes to the substrate specificity of aminopeptidase A
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DOI：
10.1021/bi9925726
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发表时间：
2000-03-21
期刊：
影响因子：
2.9
通讯作者：
Llorens-Cortès, C
中科院分区：
文献类型：
--
作者：
Iturrioz, X;Vazeux, G;Llorens-Cortès, C
氨肽酶A（EC 3.4.11.7，阿帕）是一种130 kDa的膜结合蛋白酶，其含有在锌金属蛋白酶家族锌蛋白中发现的HEXXH共有序列。除了具有催化作用的锌原子外，阿帕还含有一个Ca 2+离子，可以增加其酶活性。比对小鼠阿帕、APN和其他单锌氨肽酶的序列，鉴定出保守的组氨酸（小鼠阿帕中的His 450）。用苯丙氨酸（Phe 450）取代该残基，通过定点诱变导致阿帕活性水平明显降低，阿帕对Ca 2+的敏感性发生变化（野生型中Ca 2+的EC 50为25 μ M，突变体中仅为279 μ M）。动力学研究，与一个超大的Ca 2+浓度（4 mM），表明K-m的突变体酶的底物α-L-谷氨酰-β-萘酰胺是25倍高于野生型，而k（猫）值低得多（22倍）。因此，总的来说，野生型酶具有比突变体高571倍的切割效率。两种不同类型的抑制剂，谷氨酸硫醇和谷氨酸膦酸盐化合物的抑制效力，显着降低（因子19和22，分别）突变的酶比野生型酶。相反，赖氨酸硫醇的抑制作用不受影响。这些数据有力地表明，His 450是催化活性的关键，并通过与底物的P1羧酸酯侧链的相互作用参与底物结合。此外，His 450与Ca 2+一起可能有助于阿帕对N末端酸性氨基酸残基的底物特异性。
Aminopeptidase A (EC 3.4.11.7, APA) is a 130 kDa membrane-bound protease that contains the HEXXH consensus sequence found in the zinc metalloprotease family, the zincins. In addition to the catalytic zinc atom, APA contains a Ca2+ ion that increases its enzymatic activity. Aligning the sequences of the mouse APA, APN, and other monozinc aminopeptidases led to the identification of a conserved histidine (His 450 in mouse APA). Replacing this residue with a phenylalanine (Phe 450) by site-directed mutagenesis resulted in markedly lower levels of APA activity and in a change in the sensitivity of APA to Ca2+ (the EC50 for Ca2+ was 25 mu M in the wild type and only 279 mu M in the mutant). Kinetic studies, with a supramaximal Ca2+ concentration (4 mM), showed that the K-m of the mutant enzyme for the substrate alpha-L-glutamyl-beta-naphthylamide was 25 times higher than that of the wild type, whereas the k(cat) value was much lower (factor of 22). Thus, overall, the wild-type enzyme had a cleavage efficiency that was 571 times higher than that of the mutant. The inhibitory potencies of two different classes of inhibitors, a glutamate thiol and a glutamate phosphonate compound, were significantly lower (factors of 19 and 22, respectively) for the mutated enzyme than for the wild-type enzyme. In contrast, inhibition by lysine thiol was unaffected. These data strongly suggest that His 450 is critical for catalytic activity and is involved in substrate binding via interaction with the P1 carboxylate side chain of the substrate. Furthermore, His 450, together with Ca2+, may contribute to the substrate specificity of APA for N-terminal acidic amino acid residues.