The role of the C-terminal region in phosphoglycerate mutase

Abstract

Removal of the C-terminal seven residues from phosphoglycerate mutase from Saccharomyces cerevisiae by limited proteolysis is associated with loss of mutase activity, but no change in phosphatase activity. The presence of the cofactor 2, 3-bisphosphoglycerate, or of the cofactor and substrate 3-phosphoglycerate together, confers protection against proteolysis. The substrate alone offers no protection. Replacement of either or both of the two lysines at the C-terminus by glycines has only limited effects on the kinetic properties of phosphoglycerate mutase, indicating that these residues are unlikely to be involved in crucial electrostatic interactions with the substrate, intermediate or product in the reaction. However, the double-mutant form of the enzyme is more sensitive to proteolysis and is no longer protected against proteolysis by the presence of cofactor. The proteolysed wild-type and two of the mutated forms of the enzyme show a reduced response to 2-phosphoglycollate, which enhances the instability of the phospho form of the native enzyme. The phosphoglycerate mutase from Schizosaccharomyces pombe, which lacks the analogous C-terminal tail, has an inherently lower mutase activity and is also less responsive to stimulation by 2-phosphoglycollate. It is proposed that the C-terminal region of phosphoglycerate mutase helps to maintain the enzyme in its active phosphorylated form and assists in the retention of the bisphosphoglycerate intermediate at the active site. However, its role seems not to be to contribute directly to ligand binding, but rather to exert indirect effects on the transfer of the phospho group between substrate, enzyme, intermediate and product.

Regulation of Glycolytic Enzyme Phosphoglycerate Mutase-1 by Sirt1 Protein-mediated Deacetylation

“…Biochemical data suggest that this cap protects the phosphohistidine intermediate and prevents hydrolysis, which would short circuit the catalytic cycle of the mutase reaction (32,33). The C terminus of PGAM1 was hypothesized to interact with a phosphoglycerate substrate and position the substrate for catalysis (33). Supporting the important function of the cap, removing the C-terminal tail of PGAM1 in Saccharomyces cerevisiae decreased 10-fold the conversion of 3-PGA to 2-PGA (33), whereas the phosphatase activity (hydrolysis of the phosphohistidine intermediate) for this mutant was increased.…”

“…Although PGAM structures are available, the C terminus is not visible, preventing direct structural insight. Biochemical data suggest that this cap protects the phosphohistidine intermediate and prevents hydrolysis, which would short circuit the catalytic cycle of the mutase reaction (32,33). The C terminus of PGAM1 was hypothesized to interact with a phosphoglycerate substrate and position the substrate for catalysis (33).…”

“…The C terminus of PGAM1 harbors several adjacent lysine residues that include Lys-251, Lys-253, and Lys-254, and these would be invisible in the proteomic screens. This C-terminal region of PGAM has been hypothesized to affect enzyme efficiency by protecting the phosphohistidine intermediate within the activated form of the enzyme (32,33). Mutation or deletion of this region led to decreased enzyme activity and increased hydrolysis of the phosphohistidine in the active site.…”

High Resolution Structure of the Phosphohistidine-activated Form of Escherichia coli Cofactor-dependent Phosphoglycerate Mutase

“…S. pombe dPGM is typical of a group of "short" dPGMs (also including Zymomonas mobilis and Haemophilus influenzae) that have no C-terminal tail. Limited proteolysis of S. cerevisiae dPGM, removing the C-terminal seven residues, produces a protein of similar character to S. pombe dPGM with markedly reduced mutase activity and enhanced phosphatase activity (30). Most of the residues involved in interactions that hold the tail in place are conserved among all "full-length" dPGMs and link, via one or two residues, directly to the substrate binding region.…”

Analysis of the Function of a Putative 2,3-Diphosphoglyceric Acid-Dependent Phosphoglycerate Mutase from Bacillus subtilis

“…The lack of or relatively low PGM activity of YhfR is somewhat surprising, given the sequence homology of this protein with dPGMs including the enzyme from S. cerevisiae noted above. However, the yeast enzyme has 36 internal residues not present in YhfR, and the sequences in the carboxy-terminal regions of the two proteins are very different; there are also a variety of data indicating that the carboxy-terminal regions of dPGMs are important for enzyme function (18,24,25,27). In addition, comparison of putative dPGM sequences from Bacillus stearothermophilus, B. subtilis, Clostridium acetobutylicum, and Clostridium difficile, all of which are spore formers, showed a surprising lack of sequence conservation, with only ϳ13% identical residues in the proteins from these four species (data not shown).…”