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Orthophosphate binding at the dimer interface of Corynebacterium callunae starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme.

Mueller M, Nidetzky B - BMC Biochem. (2010)

Bottom Line: While the mutations affected neither content of pyridoxal 5'-phosphate cofactor nor specific activity in phosphorylase preparations as isolated, they disrupted (Thr28-->Ala, Arg141-->Ala) or decreased (Lys31-->Ala, Ser174-->Ala) the unusually strong protective effect of orthophosphate (10 or 100 mM) against inactivation at 45 degrees C and subunit dissociation enforced by imidazole, as compared to wild-type enzyme.The molecular strategy exploited for quaternary structure stabilization is to our knowledge novel among dimeric proteins.It can be distinguished clearly from the co-solute effect of orthophosphate on protein thermostability resulting from (relatively weak) interactions of the ligand with protein surface residues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.

ABSTRACT

Background: Orthophosphate recognition at allosteric binding sites is a key feature for the regulation of enzyme activity in mammalian glycogen phosphorylases. Protein residues co-ordinating orthophosphate in three binding sites distributed across the dimer interface of a non-regulated bacterial starch phosphorylase (from Corynebacterium callunae) were individually replaced by Ala to interrogate their unknown function for activity and stability of this enzyme.

Results: While the mutations affected neither content of pyridoxal 5'-phosphate cofactor nor specific activity in phosphorylase preparations as isolated, they disrupted (Thr28-->Ala, Arg141-->Ala) or decreased (Lys31-->Ala, Ser174-->Ala) the unusually strong protective effect of orthophosphate (10 or 100 mM) against inactivation at 45 degrees C and subunit dissociation enforced by imidazole, as compared to wild-type enzyme. Loss of stability in the mutated phosphorylases appeared to be largely due to weakened affinity for orthophosphate binding. Binding of sulphate mimicking the crystallographically observed "non-covalent phosphorylation" of the phosphorylase at the dimer interface did not have an allosteric effect on the enzyme activity.

Conclusions: The phosphate sites at the subunit-subunit interface of C. callunae starch phosphorylase appear to be cooperatively functional in conferring extra kinetic stability to the native dimer structure of the active enzyme. The molecular strategy exploited for quaternary structure stabilization is to our knowledge novel among dimeric proteins. It can be distinguished clearly from the co-solute effect of orthophosphate on protein thermostability resulting from (relatively weak) interactions of the ligand with protein surface residues.

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Dissociation of enzyme subunits in R141A enforced by imidazole. R141A as isolated is a mixture consisting of the native dimer and a small amount of a tetrameric form that is also active. The N-terminal His-tag causes the tetramerization [15]. The absorbance traces are in arbitrary units (a.u.). Elution profiles are shown for R141A prior to (dashed line) and after incubation in the presence of 0.4 M imidazole for 30 min (dotted line), 60 min (solid line) and 140 min (dashed-dotted line). The observed peaks correspond to tetrameric (t; 362 kDa), dimeric (d; 181 kDa) and monomeric (m; 90.6 kDa) forms of the protein. A high-molecular mass peak is also visible in some traces, presumably showing soluble aggregated protein. Loss of phosphorylase activity in the presence of imidazole is correlated with the extent to which monomer formation had occurred (data not shown). Note that all samples contained the same protein concentration (0.4 mg/ml) prior to the incubation with imidazole. The decrease in peak area for the eluted protein forms as the incubation time in the presence of imidazole increased probably reflects loss of protein due to aggregation. Insoluble aggregates are removed by centrifugation prior to gel filtration. The protein concentration of the sample applied to the Superdex column was not measured.
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Figure 3: Dissociation of enzyme subunits in R141A enforced by imidazole. R141A as isolated is a mixture consisting of the native dimer and a small amount of a tetrameric form that is also active. The N-terminal His-tag causes the tetramerization [15]. The absorbance traces are in arbitrary units (a.u.). Elution profiles are shown for R141A prior to (dashed line) and after incubation in the presence of 0.4 M imidazole for 30 min (dotted line), 60 min (solid line) and 140 min (dashed-dotted line). The observed peaks correspond to tetrameric (t; 362 kDa), dimeric (d; 181 kDa) and monomeric (m; 90.6 kDa) forms of the protein. A high-molecular mass peak is also visible in some traces, presumably showing soluble aggregated protein. Loss of phosphorylase activity in the presence of imidazole is correlated with the extent to which monomer formation had occurred (data not shown). Note that all samples contained the same protein concentration (0.4 mg/ml) prior to the incubation with imidazole. The decrease in peak area for the eluted protein forms as the incubation time in the presence of imidazole increased probably reflects loss of protein due to aggregation. Insoluble aggregates are removed by centrifugation prior to gel filtration. The protein concentration of the sample applied to the Superdex column was not measured.

Mentions: Assay conditions were used which in the wild-type phosphorylase lead to formation of a stable, monomeric apo-enzyme [11]. The presence of the His-tag does not alter the denaturation behaviour of the native CcGlgP in the presence of imidazole. Loss of enzyme activity in the assay reflects dissociation of the protein subunits and is partly reversible upon addition of external PLP [11]. Results of time course experiments comparing wild-type enzyme and the various mutants revealed that the process of inactivation in each enzyme was kinetically first-order (data not shown), and Table 2 summarizes t1/2 values for denaturation by imidazole in the absence and presence of 5.0 mM orthophosphate. The stability of the wild-type enzyme was slightly lower than reported previously which is explicable on account of a 6-fold lower protein concentration used in the experiments described herein, as compared to literature [11]. It was confirmed that addition of PLP restored activity in partly inactivated enzyme preparations, and time-dependent conversion of the native R141A dimer into an inactive monomer was demonstrated by using size exclusion chromatography (Figure 3).


Orthophosphate binding at the dimer interface of Corynebacterium callunae starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme.

Mueller M, Nidetzky B - BMC Biochem. (2010)

Dissociation of enzyme subunits in R141A enforced by imidazole. R141A as isolated is a mixture consisting of the native dimer and a small amount of a tetrameric form that is also active. The N-terminal His-tag causes the tetramerization [15]. The absorbance traces are in arbitrary units (a.u.). Elution profiles are shown for R141A prior to (dashed line) and after incubation in the presence of 0.4 M imidazole for 30 min (dotted line), 60 min (solid line) and 140 min (dashed-dotted line). The observed peaks correspond to tetrameric (t; 362 kDa), dimeric (d; 181 kDa) and monomeric (m; 90.6 kDa) forms of the protein. A high-molecular mass peak is also visible in some traces, presumably showing soluble aggregated protein. Loss of phosphorylase activity in the presence of imidazole is correlated with the extent to which monomer formation had occurred (data not shown). Note that all samples contained the same protein concentration (0.4 mg/ml) prior to the incubation with imidazole. The decrease in peak area for the eluted protein forms as the incubation time in the presence of imidazole increased probably reflects loss of protein due to aggregation. Insoluble aggregates are removed by centrifugation prior to gel filtration. The protein concentration of the sample applied to the Superdex column was not measured.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837607&req=5

Figure 3: Dissociation of enzyme subunits in R141A enforced by imidazole. R141A as isolated is a mixture consisting of the native dimer and a small amount of a tetrameric form that is also active. The N-terminal His-tag causes the tetramerization [15]. The absorbance traces are in arbitrary units (a.u.). Elution profiles are shown for R141A prior to (dashed line) and after incubation in the presence of 0.4 M imidazole for 30 min (dotted line), 60 min (solid line) and 140 min (dashed-dotted line). The observed peaks correspond to tetrameric (t; 362 kDa), dimeric (d; 181 kDa) and monomeric (m; 90.6 kDa) forms of the protein. A high-molecular mass peak is also visible in some traces, presumably showing soluble aggregated protein. Loss of phosphorylase activity in the presence of imidazole is correlated with the extent to which monomer formation had occurred (data not shown). Note that all samples contained the same protein concentration (0.4 mg/ml) prior to the incubation with imidazole. The decrease in peak area for the eluted protein forms as the incubation time in the presence of imidazole increased probably reflects loss of protein due to aggregation. Insoluble aggregates are removed by centrifugation prior to gel filtration. The protein concentration of the sample applied to the Superdex column was not measured.
Mentions: Assay conditions were used which in the wild-type phosphorylase lead to formation of a stable, monomeric apo-enzyme [11]. The presence of the His-tag does not alter the denaturation behaviour of the native CcGlgP in the presence of imidazole. Loss of enzyme activity in the assay reflects dissociation of the protein subunits and is partly reversible upon addition of external PLP [11]. Results of time course experiments comparing wild-type enzyme and the various mutants revealed that the process of inactivation in each enzyme was kinetically first-order (data not shown), and Table 2 summarizes t1/2 values for denaturation by imidazole in the absence and presence of 5.0 mM orthophosphate. The stability of the wild-type enzyme was slightly lower than reported previously which is explicable on account of a 6-fold lower protein concentration used in the experiments described herein, as compared to literature [11]. It was confirmed that addition of PLP restored activity in partly inactivated enzyme preparations, and time-dependent conversion of the native R141A dimer into an inactive monomer was demonstrated by using size exclusion chromatography (Figure 3).

Bottom Line: While the mutations affected neither content of pyridoxal 5'-phosphate cofactor nor specific activity in phosphorylase preparations as isolated, they disrupted (Thr28-->Ala, Arg141-->Ala) or decreased (Lys31-->Ala, Ser174-->Ala) the unusually strong protective effect of orthophosphate (10 or 100 mM) against inactivation at 45 degrees C and subunit dissociation enforced by imidazole, as compared to wild-type enzyme.The molecular strategy exploited for quaternary structure stabilization is to our knowledge novel among dimeric proteins.It can be distinguished clearly from the co-solute effect of orthophosphate on protein thermostability resulting from (relatively weak) interactions of the ligand with protein surface residues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.

ABSTRACT

Background: Orthophosphate recognition at allosteric binding sites is a key feature for the regulation of enzyme activity in mammalian glycogen phosphorylases. Protein residues co-ordinating orthophosphate in three binding sites distributed across the dimer interface of a non-regulated bacterial starch phosphorylase (from Corynebacterium callunae) were individually replaced by Ala to interrogate their unknown function for activity and stability of this enzyme.

Results: While the mutations affected neither content of pyridoxal 5'-phosphate cofactor nor specific activity in phosphorylase preparations as isolated, they disrupted (Thr28-->Ala, Arg141-->Ala) or decreased (Lys31-->Ala, Ser174-->Ala) the unusually strong protective effect of orthophosphate (10 or 100 mM) against inactivation at 45 degrees C and subunit dissociation enforced by imidazole, as compared to wild-type enzyme. Loss of stability in the mutated phosphorylases appeared to be largely due to weakened affinity for orthophosphate binding. Binding of sulphate mimicking the crystallographically observed "non-covalent phosphorylation" of the phosphorylase at the dimer interface did not have an allosteric effect on the enzyme activity.

Conclusions: The phosphate sites at the subunit-subunit interface of C. callunae starch phosphorylase appear to be cooperatively functional in conferring extra kinetic stability to the native dimer structure of the active enzyme. The molecular strategy exploited for quaternary structure stabilization is to our knowledge novel among dimeric proteins. It can be distinguished clearly from the co-solute effect of orthophosphate on protein thermostability resulting from (relatively weak) interactions of the ligand with protein surface residues.

Show MeSH
Related in: MedlinePlus