Abstract
The enzymatic activities catalyzed by bisphosphoglycerate mutase (BPGM, EC 5.4.2.4) have been shown to occur at a unique active site, with distinct binding sites for diphosphoglycerates and monophosphoglycerates. The physiological phosphatase activator (2-phosphoglycolate) binds to BPGM at an undetermined site. BPGM variants were constructed by site-directed mutagenesis of three amino acid residues in the active site to identify residues specifically involved in the binding of the monophosphoglycerates and 2-phosphoglycolate. Substitution of Cys22 by functionally conservative residues, Thr or Ser, caused a great decrease in 2-phosphoglycolate-stimulated phosphatase activity and in the Ka value of the activator, whereas it caused no change in other catalytic activities or in the Km values of 2,3-diphosphoglycerate (2,3-DPG) and glycerate 3-phosphate (3-PG, EC 1.1.1.12), indicating that Cys22 is specifically involved either directly or indirectly in 2-phosphoglycolate binding. Kinetic experiments showed that the Ka of the cofactor and the Km of 3-PG were affected by the substitution of Ser23 indicating that this residue is necessary for the fixation of both 3-PG and 2-phosphoglycolate. The R89K variant has previously been shown to have a modified Km value for monophosphoglycerates, however, its affinity for 2-phosphoglycolate is unaltered, suggesting that Arg89 is specifically involved in monophosphoglycerates binding. CD spectroscopic studies of substrates and cofactor binding showed that 2,3-DPG induced structural modifications of normal and mutated enzymes which could be due to protein phosphorylation. Addition of 2-phosphoglycolate to phosphorylated proteins with normal affinity for the cofactor produced spectra with the same characteristics as unphosphorylated species. In summary, monophosphoglycerates and 2-phosphoglycolate have partially distinct binding sites in human BPGM. The specific implication of the Cys22 residue in 2-phosphoglycolate binding is of great significance in the design of analogs of therapeutic benefit.
Highlights
BPGM variants were constructed by site-directed mutagenesis of three amino acid residues in the active site to identify residues involved in the binding of the monophosphoglycerates and 2-phosphoglycolate
Purification of Wild-type and Mutant BPGMs Expressed in E. coli—In the human, murine, and rabbit BPGMs, the Cys22 and Ser23 residues are strictly conserved (Table I), at the position of Ser23 in BPGMs there is always a Gly in the MPGMs from human muscle and brain and from yeast
BPGM, rabbit BPGM, murine BPGM, human MPGM type M (MPGM-M), human MPGM type B (MPGM-B), and yeast MPGM Residue numbers refer to the human BPGM amino acid sequence
Summary
BPGM variants were constructed by site-directed mutagenesis of three amino acid residues in the active site to identify residues involved in the binding of the monophosphoglycerates and 2-phosphoglycolate. Substitution of Cys by functionally conservative residues, Thr or Ser, caused a great decrease in 2-phosphoglycolate-stimulated phosphatase activity and in the Ka value of the activator, whereas it caused no change in other catalytic activities or in the Km values of 2,3diphosphoglycerate (2,3-DPG) and glycerate 3-phosphate (3-PG, EC 1.1.1.12), indicating that Cys is involved either directly or indirectly in 2-phosphoglycolate binding. CD spectroscopic studies of substrates and cofactor binding showed that 2,3-DPG induced structural modifications of normal and mutated enzymes which could be due to protein phosphorylation. Addition of 2-phosphoglycolate to phosphorylated proteins with normal affinity for the cofactor produced spectra with the same characteristics as unphosphorylated species. BPGM displays a mutase reaction similar to that of the glycolytic enzyme monophosphoglycerate mutase (MPGM, EC 5.4.2.1) which reversibly converts glycerate 3-phosphate (3-PG) to glycerate 2-phosphate [5, 6]
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