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.

Isolation and characterization of the human 2,3-bisphosphoglycerate mutase gene.

The human 2,3-bisphosphoglycerate mutase gene was isolated from genomic libraries and analyzed by Southern blots and DNA sequencing. The transcription initiation site was localized by primer extension as well as by S1 protection of the mRNA. The gene extends over 22 kilobase pairs; it is composed of two introns (8.8 and 11.5 kilobase pairs long) and three exons (84, 662, and 965 base pairs long). The second exon correlates with a functional subdomain of the protein, as shown by comparison with the yeast phosphoglycerate mutase structure. The sequence TAGAAAA was found 30 bases upstream from the transcription initiation site and could be analogous to the TATA box. A sequence homologous to the CCAAT box was found twice, at positions -75 and -178. There is no GC-rich sequence or GC box in the 5'-flanking region of the gene. Northern blot analysis indicates that the 2,3-bisphosphoglycerate mutase mRNA is detected mainly in erythroid tissues and cell lines, although it is also present in low amounts in a nonerythroid tissue. A comparison of the 5'-upstream sequences with other promoters active only in erythroid cells did not reveal any common signal that could be responsible for the "erythroid promoter."

Molecular cloning and nucleotide sequence of murine 2,3-bisphosphoglycerate mutase cDNA.

Cloning and sequencing of a murine cDNA with the entire coding region of 2,3-bisphosphoglycerate mutase is reported, as a prerequisite for further expression studies of this erythroid specific enzyme in Friend mouse erythroleukemia cells. A comparison between species of the deduced amino acid sequences of these proteins shows 20 substitutions between mouse and human and 21 between mouse and rabbit: none of these substitutions are in positions assumed to be in the active site. Amino acid alignment with the other related enzymes, the phosphoglycerate mutases, in combination with crystallographic data from yeast phosphoglycerate mutase, gives some insight into the structure/function correlation for this protein family. Amino acid residues which are most likely critical for either 2,3-bisphosphoglycerate mutase or phosphoglycerate mutase function are pointed out. Concerning the phylogenetic analysis, phosphoglycerate mutases B and M from mammalians appear to have diverged with the yeast enzyme from a common ancestor, before the emergence of the 2,3-bisphosphoglycerate mutases.