title: Critical role of human bisphosphoglycerate mutase Cys22 in the phosphatase activator-binding site.
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.
authors.affiliation: INSERM U.91, Hôpital Henri Mondor, 94010 Créteil, France.
authors.forename: P
authors.initials: P
authors.lastname: Ravel
authors.forename: C T
authors.initials: CT
authors.lastname: Craescu
authors.forename: N
authors.initials: N
authors.lastname: Arous
authors.forename: J
authors.initials: J
authors.lastname: Rosa
authors.forename: M C
authors.initials: MC
authors.lastname: Garel
mesh_terms: D001665:Binding Sites; D001731:Bisphosphoglycerate Mutase / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D003545:Cysteine; D004926:Escherichia coli; D006016:Glycolates / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D006016:Glycolates; D010744:Phosphoric Monoester Hydrolases; D001731:Bisphosphoglycerate Mutase; C013193:phosphoglycolate; D003545:Cysteine