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-Dglycerate 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 Arg'-His"'-Gly"1-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 Gly'3 with an Arg or a Lys. In another experiment, we replaced Gly'3 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (n-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly'3 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly'3 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.Some enzymes displaying phosphatase activities such as human prostatic acid phosphatase (1, 2), lysosomal (3) and yeast (4) acid phosphatases, erythrocyte bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4) (5-7), glycolytic phosphoglycerate mutase (PGM; D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) (7,8), and hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (fructose-2,6-bisphosphate 2-phosphatase; D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 3.1.3.46) (1) possess a His residue in their active site, which is transiently phosphorylated during the course of the phosphoryl transfer. It was suggested that most such enzymes involved in the binding of phosphate esters should have at least one cationic group suitably disposed near the active site His in order to facilitate the ori...