Screening of activators of 2,3-diphosphoglycerate mutase from traditional Chinese herb medicines.

OBJECTIVE: To screen activators of 2,3-diphosphoglycerate (BPG) mutase (BPGM) from Chinese herb medicines, so as to improve the hypoxia tolerance of erythrocytes. METHODS: BPGM was used as the receptor and Chinese medicine ingredients database was used as the ligand in the study. After Lipinski rule of five screening, LibDock and CDOCKER docking were used for virtual screening. The effect of the screened compounds on the affinity of BPGM in erythrocytes was verified. Finally, the erythrocytes were incubated in vitro to establish the erythrocyte hypoxia model, and the effect of the compound on the activity of BPGM in the erythrocyte hypoxia model was verified. RESULTS: Ten compounds with highest binding affinity to BPGM were selected by LibDock and CDOCKER, and the cytoplasm protein was incubated with the ten compounds. Compared with blank control group, methyl rosmarinate group, dihydrocurcumin high dose group, octahydrocurcumin medium dose group and coniferyl ferulate high dose group were able to further activate the BPGM, significantly increase the levels of 2, 3-BPG in normal erythrocytes (all P<0.05); while the low dose of tetrahydrocurcumin, high dose and low dose of aurantiamide, hexahydrocurcumin and medium dose of N- (p-coumaroyl) serotonin had a tendency to increase the contents of 2,3-BPG in normal erythrocytes (all P>0.05). In the hypoxic red blood cells, the medium dose methyl rosmarinate, medium dose octahydrocurcumin, high dose hexahydrocurcumin and medium dose N-(p-coumaroyl) serotonin could significantly increase the contents of 2,3-BPG (all P<0.05). CONCLUSION: The methyl rosmarinate, octahydrocurcumin, hexahydrocurcumin and N-(p-coumaroyl) serotonin could activate BPGM and increase the contents of 2,3-BPG in hypoxic erythrocytes.

Crystal structure of human bisphosphoglycerate mutase.

Bisphosphoglycerate mutase is a trifunctional enzyme of which the main function is to synthesize 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. The gene coding for bisphosphoglycerate mutase from the human cDNA library was cloned and expressed in Escherichia coli. The protein crystals were obtained and diffract to 2.5 A and produced the first crystal structure of bisphosphoglycerate mutase. The model was refined to a crystallographic R-factor of 0.200 and R(free) of 0.266 with excellent stereochemistry. The enzyme remains a dimer in the crystal. The overall structure of the enzyme resembles that of the cofactor-dependent phosphoglycerate mutase except the regions of 13-21, 98-117, 127-151, and the C-terminal tail. The conformational changes in the backbone and the side chains of some residues reveal the structural basis for the different activities between phosphoglycerate mutase and bisphosphoglycerate mutase. The bisphosphoglycerate mutase-specific residue Gly-14 may cause the most important conformational changes, which makes the side chain of Glu-13 orient toward the active site. The positions of Glu-13 and Phe-22 prevent 2,3-bisphosphoglycerate from binding in the way proposed previously. In addition, the side chain of Glu-13 would affect the Glu-89 protonation ability responsible for the low mutase activity. Other structural variations, which could be connected with functional differences, are also discussed.

Isolation, characterization, and structure of a mutant 89 Arg----Cys bisphosphoglycerate mutase. Implication of the active site in the mutation.

Bisphosphoglycerate mutase (EC 5.4.2.4.) is a trifunctional enzyme which displays synthase, mutase, and phosphatase activities. The purification, characterization, and structural study of an abnormal form of the enzyme, isolated from a patient which we reported earlier (Rosa, R., Prehu, M. O., Beuzard, Y., and Rosa, J. (1978) J. Clin. Invest. 62, 907-915), is described. The abnormal enzyme, present at 50% of the level of the normal enzyme as estimated by immunological methods, showed elevated electrophoretic mobility and hybridized with erythrocyte phosphoglycerate mutase (EC 5.4.2.1.) in the same manner as the normal control. The mutant enzyme was unstable at 55 degrees C and could be protected against thermal instability by 0.5 mM glycerate 2,3-bisphoshate but not by either glycerate 3-phosphate or glycolate 2-phosphate. Two of the three functions of the mutant enzyme were distinct from those of the normal protein. The specific activity of the synthase was 0.57% of normal and that of the mutase 4.1%. By contrast, the specific phosphatase activity was not affected by the mutation. However, the phosphatase activity of the mutated protein was markedly less stimulated by glycolate-2-phosphate than that of the control. High performance liquid chromatography analysis of tryptic peptides derived from the mutant enzyme showed an abnormal profile with the absence of two peaks normally containing the T12 and T13 peptides and without the appearance of a supplementary peak. Amino acid sequence and mass spectrometric analysis demonstrated the substitution of Arg----Cys residue in position 89 producing an uncleaved T12-T13 present in the same peak as the T6. Considered together, our data suggest that Arg-89 is located at or near the active site of bisphosphoglycerate mutase and that this residue is probably involved in the binding of monophosphoglycerates.

Sequential changes in red cell glycolytic enzymes and intermediates and possible control mechanisms in the first two months of postnatal life in lambs.

The sequential changes in several glycolytic enzymes, glucose-6-phosphate dehydrogenase, glycolytic intermediates, and adenosine triphosphate, as well as intracellular pH and plasma inorganic phosphorus were followed simultaneously in eight lambs from birth to 2 months of age. The activities of all glycolytic enzymes and glucose-6-phosphate dehydrogenase were elevated at birth. The 2,3-diphosphoglycerate concentration increased markedly postnatally and was associated with a simultaneous increase in the concentrations of red cell glucose-6-phosphate and total triose phosphate and a decrease in intracellular pH. Inorganic phosphorus also increased and correlated with the 2,3-diphosphoglycerate concentration in the first 10 days of postnatal life. The content of red cell 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and ATP increased slightly. These results suggested increased glycolytic flux through the diphosphoglycerate mutase reaction which resulted in net synthesis of 2,3-diphosphoglycerate. The red cell total triose phosphate peaked and fell initially, followed by glucose-6-phosphate and 2,3-diphosphoglycerate suggesting inhibition of phosphofructokinase activity and a decrease in glycolysis secondary to decreased red cell intracellular pH. After 10 days of postnatal life all glycolytic intermediates fell simultaneously, which correlated with a decrease in activity of the glycolytic enzymes.