Amino acid residues involved in the catalytic site of human erythrocyte bisphosphoglycerate mutase

Abstract

Human bisphosphoglycerate mutase (Grip, mutase) is a trifunctional enzyme which synthesizes and degrades Grip, in red cells. Among the amino acid residues involved in its active site there are two conserved histidine residues, HislO which is phosphorylated during the catalytic process and His187 for which only speculative data have been made about the potential role during the reactions. Another amino acid residue, Arg89, had not been described as part of this active site but we have recently shown that a natural mutant Arg89+Cys was highly thermolabile and showed severe perturbations of its enzymatic properties. To understand better the exact role of these residues, replacements of HislO by Gly (HlOG) or Asp (HlOD), His187 by Asn (H187N), Tyr (H187Y) or Asp (H187D) and Arg89 by Cys (R89C), Ser (R89S), Gly (R89G) or Lys (R89K) were performed by site-directed mutagenesis. The results obtained in this report show that replacement of the HislO residue completely abolished the enzymatic activities. Concerning the Hisl 87 residue, our results afford arguments that it plays an essential role in the three catalytic activities. Indeed all these activities are abolished in the two H187Y and H187D variants, whereas they are detectable though strongly diminished, for the H187N variant. In addition mutations at Hisl 87 could be distinguishable from those at HislO since the former resulted in a thermolabile enzyme, whereas no significant change in heat stability was observed for the latter. It is noteworthy that the H187N variant is protected against thermal instability by glycerate 2,3-bisphosphate (Grip,). Concerning the Arg89 mutants, R89C, R89S and R89G, the three variants showed characteristics identical to those found in the natural R89C mutant, i.e. loss of 99% of synthase activity, consistent decrease of mutase and 2-phosphoglycolate-stimulated phosphatase activities whereas the unstimulated phosphatase activity was normal. Moreover these mutants were unstable at 55°C but Grip, was able to protect them against thermal instability. In contrast, the R89K mutant was stable at 55°C. Its synthase and unstimulated phosphatase activities were normal but its mutase and 2-phosphoglycolate-stimulated phosphatase activities were decreased. In addition, K,,, values for monophosphoglycerates were increased (3.2-fold) in the synthase but normal in mutase activities, whereas K,values for GriP, were normal in mutase and phosphatase activities. In conclusion, all these results afford evidence that the His187 residue contributes to both the conformational stability and the three catalytic activities of the enzyme, while HislO is essential for all the enzymatic reactions; the presence of a basic residue at position 89 is necessary to maintain the enzyme in a functional structure since this residue is involved, either directly or indirectly, in the binding of monophosphoglycerates and 2-phosphoglycolate to the enzyme.

Critical Role of Human Bisphosphoglycerate Mutase Cys22 in the Phosphatase Activator-binding Site

“…As previously reported (24), the R89K mutated protein presents increased Michaelis constants for monophosphoglycerates, whereas the 2,3-DPG one is not affected. Our present results demonstrated that its affinity for 2-phosphoglycolate was also unchanged, indicating that Arg 89 is only involved, directly or indirectly, in the monophosphoglycerate-binding sites.…”

“…Arg 89 Is Specifically Involved in the Monophosphoglyceratebinding Site-Previous results obtained in our laboratory strongly suggested that the Arg 89 residue of human BPGM is specifically involved in the 3-PG-binding site (24,27). We completed the functional studies on the R89K mutant by investigating the ion-stimulated phosphatase activity and measuring the affinity constant for 2-phosphoglycolate.…”

“…A prokaryotic expression vector constructed in our laboratory (23) was used to synthesize wild-type and mutant forms of BPGM in Escherichia coli. Analysis of the catalytic properties of variants have shown that Arg 89 is specifically involved in the monophosphoglycerate-binding sites (24) as has been reported for a natural human BPGM variant (25)(26)(27). In contrast, we have shown that Gly 13 is specifically involved in the diphosphoglycerate-binding sites and that replacement of Gly 13 by a positively charged amino acid residue greatly activates the phosphatase reaction, whereas the synthase and mutase reactions are greatly reduced (28).…”

“…The native and mutated enzymes were purified by an established procedure (23,33), however, the lysate was not heated prior to chromatography and the first purification step was on an high pressure liquid chromatography column of Fractogel TSK-AF blue at room temperature (24). The purified enzymes were stored in sodium phosphate buffer containing 20% glycerol at Ϫ80°C until used.…”

“…Further enzymatic studies on the variant R89K described previously (24) are also reported. Circular dichroism spectroscopy was used to study modifications in the secondary structure of the purified proteins, both alone and in the presence of substrates or cofactor.…”