Bueso J, Cervera J, Fresquet V, Marina A, Lusty C J, Rubio V
Instituto de Investigaciones Citológicas (FVIB), Instituto de Biomedicina de Valencia (CSIC), Spain.
Biochemistry. 1999 Mar 30;38(13):3910-7. doi: 10.1021/bi982871f.
Photoaffinity labeling with IMP was used to attach covalently this activator to its binding site of Escherichia coli carbamoyl phosphate synthetase. We now identify histidine 995 of the large enzyme subunit as the amino acid that is cross-linked with IMP. The identification was carried out by comparative peptide mapping in two chromatographic systems of peptides differentially labeled with [3H]IMP and with the labeled inhibitor [14C]UMP, followed by automated Edman degradation and radiosequence analysis. Site-directed substitution of His995 by alanine confirmed His995 to be the only amino acid in the protein forming a covalent adduct with IMP. The His995Ala mutant protein was soluble and active and exhibited normal kinetics for the activator ornithine and for the substrates in the presence of ornithine. However, the mutation selectively induced changes in the activation by IMP and the inhibition by UMP, and it abolished the photolabeling of the enzyme by IMP without affecting the photolabeling by the inhibitor UMP. Since UMP is cross-linked to Lys993 [Cervera, J., et al. (1996) Biochemistry 35, 7247-7255] only two residues upstream of the site of IMP labeling, the results provide structural evidence for earlier proposals which suggested that UMP and IMP bind in a single or overlapping site. The two residues are within the region previously proposed as the binding fold for the nucleotide effectors. In the crystal structure of the enzyme, Lys993 and His995 are exposed and line a crevice where a Pi molecule was found [Thoden, J. B., et al. (1997) Biochemistry 36, 6305-6316]. UMP and IMP appear to bind in this crevice, possibly toward the C-side of the beta-sheet in a Rossman fold. Their binding in this site is consistent with the selectivity of adduct formation of UMP with Lys993 and of IMP with His995. It is also consistent with the nonessentiality of His995 for the binding, since the interactions with other residues that line the crevice must contribute a large part of the binding energy. The lack of an effect of the mutation on the activation by ornithine is consistent with the binding of this activator in a separate site in the protein.
采用 IMP 进行光亲和标记，将这种激活剂共价连接到大肠杆菌氨甲酰磷酸合成酶的结合位点上。我们现在确定大酶亚基的组氨酸 995 是与 IMP 发生交联的氨基酸。通过在两种色谱系统中对分别用 [3H]IMP 和标记抑制剂 [14C]UMP 进行差异标记的肽段进行比较肽图分析，随后进行自动 Edman 降解和放射性序列分析，完成了这一鉴定。将 His995 定点替换为丙氨酸证实 His995 是该蛋白中唯一与 IMP 形成共价加合物的氨基酸。His995Ala 突变蛋白可溶且有活性，在有鸟氨酸存在的情况下，对激活剂鸟氨酸和底物表现出正常的动力学。然而，该突变选择性地诱导了 IMP 激活和 UMP 抑制的变化，并且消除了 IMP 对该酶的光标记，而不影响抑制剂 UMP 的光标记。由于 UMP 与 Lys993 交联 [塞尔韦拉，J. 等人（1996 年）《生物化学》35 卷，7247 - 7255 页]，仅在 IMP 标记位点上游两个残基处，这些结果为早期提出的 UMP 和 IMP 结合在单个或重叠位点的提议提供了结构证据。这两个残基位于先前提出的核苷酸效应物结合折叠区域内。在该酶的晶体结构中，Lys993 和 His995 暴露在外，并排列在一个发现有 Pi 分子的裂隙处 [托登，J. B. 等人（1997 年）《生物化学》36 卷，6305 - 6316 页]。UMP 和 IMP 似乎结合在这个裂隙中，可能朝向 Rossman 折叠中 β - 折叠的 C 端。它们在这个位点的结合与 UMP 与 Lys993 以及 IMP 与 His995 形成加合物的选择性一致。这也与 His995 对结合的非必要性一致，因为与裂隙中其他残基的相互作用必定贡献了大部分结合能。该突变对鸟氨酸激活无影响，这与该激活剂在蛋白质中一个单独位点的结合一致。
