Human bisphosphoglycerate mutase. Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism.
Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin. In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. The three activities have been demonstrated to be catalysed at a unique active site. To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis. For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter. Human bisphosphoglycerate mutase produced in E. coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme. Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced. Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme. In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity. These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme.
Garel
M C
MC
Institut National de la Santé et de la Recherche Médicale Unité 91,Hôpital Henri Mondor, Créteil, France.
Joulin
V
V
Le Boulch
P
P
Calvin
M C
MC
Préhu
M O
MO
Arous
N
N
Longin
R
R
Rosa
R
R
Rosa
J
J
Cohen-Solal
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001731:Bisphosphoglycerate Mutase / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010770:Phosphotransferases / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D010770:Phosphotransferases; D001731:Bisphosphoglycerate Mutase