Structure and activity of phosphoglycerate mutase
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The structure of yeast phosphoglycerate mutase determined by X-ray crystallographic and amino acid sequence studies has been interpreted in terms of the chemical, kinetic and mechanistic observations made on this enzyme. There are two histidine residues at the active site, with imidazole groups almost parallel to each other and approximately 0.4 nm apart, positioned close to the 2 and 3 positions of the substrate. The simplest interpretation of the available information suggests that a ping-pong type mechanism operates in which at least one of these histidine residues participates in the phosphoryl transfer reaction. The flexible C-terminal region also plays an important role in the enzymic reaction.Keywords:
Phosphoglycerate mutase
Phosphoglycerate kinase
Imidazole
Mutase
Abstract Cobamides, a uniquely diverse family of enzyme cofactors related to vitamin B 12 , are produced exclusively by bacteria and archaea but used in all domains of life. While it is widely accepted that cobamide-dependent organisms require specific cobamides for their metabolism, the biochemical mechanisms that make cobamides functionally distinct are largely unknown. Here, we examine the effects of cobamide structural variation on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). The in vitro binding affinity of MCM for cobamides can be dramatically influenced by small changes in the structure of the lower ligand of the cobamide, and binding selectivity differs between bacterial orthologs of MCM. In contrast, variations in the lower ligand have minor effects on MCM catalysis. Bacterial growth assays demonstrate that cobamide requirements of MCM in vitro largely correlate with in vivo cobamide dependence. This result underscores the importance of enzyme selectivity in the cobamide-dependent physiology of bacteria.
Mutase
Phosphoglycerate mutase
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The structure of yeast phosphoglycerate mutase determined by X-ray crystallographic and amino acid sequence studies has been interpreted in terms of the chemical, kinetic and mechanistic observations made on this enzyme. There are two histidine residues at the active site, with imidazole groups almost parallel to each other and approximately 0.4 nm apart, positioned close to the 2 and 3 positions of the substrate. The simplest interpretation of the available information suggests that a ping-pong type mechanism operates in which at least one of these histidine residues participates in the phosphoryl transfer reaction. The flexible C-terminal region also plays an important role in the enzymic reaction.
Phosphoglycerate mutase
Phosphoglycerate kinase
Imidazole
Mutase
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Mutase
Sequence (biology)
Cyanogen bromide
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The glycolytic enzyme phosphoglycerate mutase exists in two evolutionarily unrelated forms. Vertebrates have only the 2,3‐bisphosphoglycerate‐dependent enzyme (dPGM), whilst higher plants have only the cofactor‐independent enzyme (iPGM). Certain eubacteria possess genes encoding both enzymes, and their respective metabolic roles and activities are unclear. We have over‐expressed, purified and characterised the two PGMs of Escherichia coli . Both are expressed at high levels, but dPGM has a 10‐fold higher specific activity than iPGM. Differential inhibition by vanadate was observed. The presence of an integral manganese ion in iPGM was confirmed by EPR spectroscopy.
Phosphoglycerate mutase
Phosphoglycerate kinase
Vanadate
Mutase
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NaOH was used to extract proteins from the cell walls of the yeast Saccharomyces cerevisiae. This treatment was shown not to disrupt yeast cells, as NaOH-extracted cells displayed a normal morphology upon electron microscopy. Moreover, extracted and untreated cells had qualitatively similar protein contents upon disruption. When yeast was grown in the presence of 1 M mannitol, two proteins were found to be present at an elevated concentration in the cell wall. These were found to be the late-embryogenic-abundant-like protein heat-shock protein 12 and the glycolytic enzyme phosphoglycerate mutase. The presence of phosphoglycerate mutase in the cell wall was confirmed by immunocytochemical analysis. Not only was the phosphoglycerate mutase in the yeast cell wall found to be active, but whole yeast cells were also able to convert 3-phosphoglycerate in the medium into ethanol, provided that the necessary cofactors were present.
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Abstract A very rapid procedure for the preparation of phosphoglycerate mutase from chicken breast muscle is presented. Commercially available frozen chicken breasts may be used. The enzyme is obtained in high yields, and the specific activity of the crystalline homogenous preparations is equal to that of the crystalline enzymes from yeast and rabbit muscle. The chicken breast enzyme requires 2,3-diphosphoglycerate for activity, and it is very similar in kinetic and molecular properties to the rabbit muscle phosphoglycerate mutase. Similarly, it also possesses 4 sulfhydryl groups per mole of enzyme. The molecular weight of the enzyme is 65,690. With the use of a gel filtration technique, it has been demonstrated that 2 moles of 2,3-diphosphoglycerate may be bound per mole of the enzyme. It appears that the binding of this phosphoglycerate may entail phosphoenzyme formation. Again, as with other pure phosphoglycerate mutases, the chicken breast muscle mutase shows some 2,3-phosphoglycerate phosphatase activity (about 1/40,000 the mutase activity).
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Phosphoglycerate mutase
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Phosphoglycerate mutase
Mutase
Phosphoglycerate kinase
Glyceraldehyde 3-phosphate dehydrogenase
Diphosphoglycerate
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The human phosphomannomutases PMM1 and PMM2 catalyze the interconversion of hexose 6-phosphates and hexose 1-phosphates. The two isoforms share 66% sequence identity and have kinetic properties similar to those of mutases in vitro but differ in their functional roles in vivo. Though the physiological role of PMM2 is catalysis of the mutase reaction that provides the mannose 1-phosphate (Man-1-P) essential for protein glycosylation, PMM1 is thought to provide a phosphohydrolase activity in the presence of inosine monophosphate (IMP), converting glucose 1,6-bisphosphate (Glu-1,6-P2) to glucose 6-phosphate (Glu-6-P), rescuing glycolysis during brain ischemia. To uncover the structural basis of how IMP binding converts PMM1 from a mutase to a phosphatase, the 1.93 Å resolution structure of PMM1 complexed with IMP was determined. The structure reveals IMP bound at the substrate recruitment site, thus inhibiting the mutase activity while simultaneously activating a phosphatase activity (IMP Kact = 1.5 μM) resulting from the hydrolysis of the phospho-enzyme. The bound structure and site-directed mutagenesis confirm that the long-range electrostatic interactions provided by Arg180 and Arg183 conserved in PMM1 are the major contributors to IMP binding, and their oblation removes phosphatase but not mutase activity. These residues are not present in the PMM2 isoform, which consequently lacks significant phosphatase activity in the presence of IMP. T2 relaxation nuclear magnetic resonance and small angle X-ray scattering together support the hypothesis that binding of IMP to PMM1 favors an enzyme conformation that is catalytically competent for water attack at the phosphoaspartyl intermediate. Such a mechanism may be generalizable to other enzymes that act through covalent intermediates.
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Phosphoglycerate mutase
Glucokinase
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