Fukasawa Kayoko M, Hirose Junzo, Hata Toshiyuki, Ono Yukio
Department of Hard Tissue Research, Graduate School of Oral Medicine, Matsumoto Dental University, School of Dentistry, Shiojiri, Nagano 399-0781, Japan.
Biochemistry. 2006 Sep 26;45(38):11425-31. doi: 10.1021/bi0604577.
Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.
氨肽酶B（EC 3.4.11.6，ApB）在体外能特异性地从肽和合成衍生物上切割N端的精氨酸或赖氨酸残基。已证明ApB具有金属肽酶家族中发现的共有序列。我们使用定点诱变确定了该酶的推定锌结合残基（His324、His328和Glu347）以及必需的Glu325残基（深泽，K.M.等人，（1999年）《生物化学杂志》339卷，497 - 502页）。为了鉴定与底物氨基端碱性氨基酸结合的残基，将编码ApB的大鼠cDNA克隆到pGEX - 4T - 3中，以便重组蛋白作为GST融合蛋白表达。使用定点诱变将ApB中的12个酸性氨基酸残基（Glu或Asp）替换为Gln或Asn。分离出这些突变体以表征其对Arg - NA的酶活性动力学参数，并与野生型ApB的参数进行比较。突变体D405N的催化效率（kcat/Km）为1.7×10⁴ M⁻¹ s⁻¹，与野生型ApB（6.2×10⁵ M⁻¹ s⁻¹）相比显著降低。将Asp405替换为Asn残基导致底物特异性发生变化，使得突变体D405N对Lys - NA的比活性是对Arg - NA的两倍（野生型ApB的情况为0.4）。此外，当将Asp405替换为Ala残基时，对于Arg - NA水解的kcat/Km比值比野生型ApB低1000倍；相反，在Tyr - NA水解中，野生型（1.1×10⁴ M⁻¹ s⁻¹）和突变型（8.2×10³ M⁻¹ s⁻¹）酶的kcat/Km比值相似。此外，将Asp - 405替换为Glu残基导致Arg - NA水解的kcat/Km比值降低6倍，而Lys - NA水解的该比值增加。然后，D405E突变体对Lys - NA水解的kcat/Km比值高于对Arg - NA水解的该比值，这与野生型ApB相反。这些数据有力地表明，Asp405残基通过与底物侧链的P1氨基相互作用参与底物结合。
