Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.
Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.
复制标题
噬菌体 lambda 终止酶 GTPase 活性的动力学特征：该酶的两个“NTPase”催化位点之间通讯的证据。
DOI：
10.1021/bi990866l
复制
发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Catalano,CE
中科院分区：
文献类型：
--
作者：
Woods,L;Catalano,CE
来自噬菌体 λ 的终止酶负责将病毒 DNA 插入衣壳内的有限空间。该酶由病毒编码的蛋白 gpA (73.3 kDa) 和 gpNu1 (20.4 kDa) 组成，分离为 gpA1·gpNu12 全酶复合物。 Lambda 终止酶具有位点特异性核酸酶活性、ATP 依赖性 DNA 链分离活性以及必须协同工作才能实现基因组包装的 ATP 酶活性。我们之前已经表征了全酶的 ATP 酶活性，并确定了每个酶亚基中的催化活性位点 [Tomka 和 Catalano (1993)Biochemistry32, 11992−11997；黄等人。 (1996)生物化学35, 2796−2803]。我们注意到 GTP 会刺激该酶的 ATP 酶活性，并且已观察到终止酶介导的 GTP 水解。这里提出的研究描述了 λ 终止酶的 GTPase 活性的动力学分析。该酶的 GTP 水解需要二价金属，在碱性 pH 值下效果最佳，并且受到盐的强烈抑制。有趣的是，虽然 GTP 在没有 DNA 的情况下可以与酶结合，但 GTP 水解严格依赖于多核苷酸的存在。与全酶两个亚基上发生的 ATP 水解不同，在 GTPase 活性的稳态动力学分析中观察到单个催化位点（kcat ≈ 37 min-1；Km ≈ 500 μM）。此外，虽然 GTP 刺激 ATP 水解（GTP 结合的表观 KD 约 135 μM），但所有检测的腺苷核苷酸都强烈抑制该酶的 GTP 酶活性。这里提供的数据表明，终止酶全酶中的两个“NTPase”催化位点进行通信，我们提出了一个描述这两个位点之间变构相互作用的模型。讨论了这种相互作用对于病毒组装所需的多个核蛋白包装复合物的组装和分解的生物学意义。
The terminase enzyme from bacteriophage λ is responsible for the insertion of viral DNA into the confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3 kDa) and gpNu1 (20.4 kDa) isolated as a gpA1·gpNu12holoenzyme complex. Lambda terminase possesses a site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activity that must work in concert to effect genome packaging. We have previously characterized the ATPase activity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka and Catalano (1993)Biochemistry32, 11992−11997; Hwang et al. (1996)Biochemistry35, 2796−2803]. We have noted that GTP stimulates the ATPase activity of the enzyme, and terminase-mediated GTP hydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activity of λ terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and is strongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTPhydrolysisis strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs at both subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis of GTPase activity (kcat≈37 min-1;Km≈ 500 μM). Moreover, while GTP stimulates ATP hydrolysis (apparentKD≈ 135 μM for GTP binding), all of the adenosine nucleotides examined strongly inhibit the GTPase activity of the enzyme. The data presented here suggest that the two “NTPase” catalytic sites in terminase holoenzyme communicate, and we propose a model describing allosteric interactions between the two sites. The biological significance of this interaction with respect to the assembly and disassembly of the multiple nucleoprotein packaging complexes required for virus assembly is discussed.