Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme
Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme
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DOI：
10.1021/bi9928905
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发表时间：
2000-07-25
期刊：
影响因子：
2.9
通讯作者：
Carmona, AK
中科院分区：
文献类型：
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作者：
Araujo, MC;Melo, RL;Carmona, AK
淬灭的荧光肽被用来研究重组野生型血管紧张素I转换酶（ACE）和两个全长突变体承载一个单一的功能活性位点（N-或C-结构域）的底物特异性的要求。我们测定了两个系列的缓激肽相关肽，即Abz-GFSPFXQ-EDDnp和Abz-GFSPFRX-EDDnp（X =天然氨基酸），其中Abz/EDDnp通过底物水解分离时出现荧光。Abz-GFSPFFQ-EDDnp优先被C结构域水解，而Abz-GFSPFQQ-EDDnp表现出更高的N结构域特异性。还合成并测定了N-乙酰基-SDKP-OH的内部淬灭荧光类似物。Abz-SDK（Dnp）P-OH，其中Abz和Dnp（2，4-二硝基苯基）是荧光供体-受体对，在D-K（Dnp）键处被ACE N-结构域（k（cat）/K-m = 1.1 μ M-1 s（-1））以高特异性切割，ACE N-结构域实际上抵抗C-结构域的水解。通过进行Abz-TDK（Dnp）P-OH和Abz-YDK（Dnp）P-OH的水解动力学显示Pt位置处含羟基氨基酸对于N结构域特异性的重要性。肽Abz-YRK（Dnp）P-OH和Abz-FRK（Dnp）P-OH被野生型ACE水解，K-m值分别为5.1和4.0 μ M，k（cat）值分别为246和210 s（-1），已被证明是ACE的优良底物。ACE的C-和N-结构域的催化特异性的分化似乎取决于底物特异性氨基酸的非常微妙的变化。一个自由的C-末端羧基基团或芳香族部分在相同的基板位置的存在下，确定与ACE活性位点，这是由氯化物调节的特定的相互作用，似乎区分两个域的活动。
Quenched fluorescence peptides were used to investigate the substrate specificity requirements for recombinant wild-type angiotensin I-converting enzyme (ACE) and two full-length mutants bearing a single functional active site (N- or C-domain). We assayed two series of bradykinin-related peptides flanked by o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp), namely, Abz-GFSPFXQ-EDDnp and Abz-GFSPFRX-EDDnp (X = natural amino acids), in which the fluorescence appeared when Abz/EDDnp are separated by substrate hydrolysis. Abz-GFSPFFQ-EDDnp was preferentially hydrolyzed by the C-domain while Abz-GFSPFQQ-EDDnp exhibits higher N-domain specificity. Internally quenched fluorescent analogues of N-acetyl-SDKP-OH were also synthesized and assayed. Abz-SDK(Dnp)P-OH, in which Abz and Dnp (2,4-dinitrophenyl) are the fluorescent donor-acceptor pair, was cleaved at the D-K(Dnp) bond with high specificity by the ACE N-domain (k(cat)/K-m = 1.1 mu M-1 s(-1)) being practically resistant to hydrolysis by the C-domain. The importance of hydroxyl-containing amino acids at the Pt position for N-domain specificity was shown by performing the kinetics of hydrolysis of Abz-TDK(Dnp)P-OH and Abz-YDK(Dnp)P-OH. The peptides Abz-YRK(Dnp)P-OH and Abz-FRK(Dnp)P-OH which were hydrolyzed by wild-type ACE with K-m values of 5.1 and 4.0 mu M and k(cat) values of 246 and 210 s(-1), respectively, have been shown to be excellent substrates for ACE. The differentiation of the catalytic specificity of the C- and N-domains of ACE seems to depend on very subtle variations on substrate-specific amino acids. The presence of a free C-terminal carboxyl group or an aromatic moiety at the same substrate position determines specific interactions with the ACE active site which is regulated by chloride and seems to distinguish the activities of both domains.