Garel M C, Arous N, Calvin M C, Craescu C T, Rosa J, Rosa R
Institut National de la Santé et de la Recherche Médicale, U.91, Hôpital Henri Mondor, Créteil, France.
Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3593-7. doi: 10.1073/pnas.91.9.3593.
To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.
迄今为止，尚未找到针对镰状细胞贫血的确切且无争议的治疗方法。镰状细胞贫血的特征是脱氧血红蛋白突变体（HbS）发生聚合，导致红细胞变形和血管阻塞并发症。由于已证明红细胞甘油酸2,3 - 二磷酸（2,3 - DPG）会促进这种聚合，一种治疗方法是通过提高双磷酸甘油酸变位酶（BPGM；3 - 磷酸 - D - 甘油酸1,2 - 磷酸变位酶，EC 5.4.2.4）的磷酸酶活性来降低红细胞内2,3 - DPG的水平。为此，我们研究了位于人BPGM活性位点序列Arg9 - His10 - Gly11 - Glu12 - Gly13中的Gly - 13的作用。该序列与远缘相关酸性磷酸酶的Arg - His - Gly - Xaa - Arg序列相似，后者催化与BPGM类似的磷酸转移，但程度更大。我们假设酸性磷酸酶序列中保守的Arg残基促进了磷酸转移。因此，在人BPGM中，我们通过定点诱变将相应的氨基酸残基Gly13替换为Arg或Lys。在另一个实验中，我们将Gly13替换为Ser，Ser是同源酵母磷酸甘油酸变位酶（D - 磷酸甘油酸2,3 - 磷酸变位酶，EC 5.4.2.1）相应位置存在的氨基酸。将Gly13突变为Ser并未改变合酶活性，而变位酶和磷酸酶活性分别增加或降低了2倍。然而，用Arg替换Gly13使磷酸酶活性提高了28.6倍，而合酶和变位酶活性降低了10倍。13位存在Lys导致磷酸酶活性的增加较小（6.5倍），但合酶和变位酶活性的降低相同。综合这些结果支持了这样的假设，即13位带正电荷的氨基酸残基，尤其是Arg，极大地激活了向水的磷酸转移。这些结果还为在酸性磷酸酶活性位点定位保守的Arg*残基并促进磷酸转移提供了依据。本文讨论了对镰状细胞病基因治疗的意义。
