Phosphoglycerate mutases from a variety of sources have been examined with the aim of defining their denaturation and renaturation (refolding) characteristics. Both cofactor (2,3-bisphosphoglycerate) dependent and independent enzymes have been examined and examples with differing quaternary configurations are included. The isolation procedures for these enzymes are reported.
The cofactor dependent enzyme from Schizosaccbaromyces pombe shows a novel monomeric configuration within this group. This configuration has been confirmed here and the amino acid analysis and partial amino acid sequence determined by Prof. J.Fothergill are reported. A moderate degree of homology to the sequence of the cofactor dependent tetrameric enzyme from bakers yeast has been demonstrated. Similarly. the degree of homology between the cofactor dependent dimeric enzyme from rabbit muscle and the bakers yeast enzyme has been examined. These studies indicate moderate relatedness.
The cofactor independent phosphoglycerate mutases exhibit a number of differences from the cofactor dependent enzymes. The divisions between these two groups have been further emphasised in an apparent metal ion requirement and a native Mr of approximately 60,000 observed for the cofactor independent enzymes examined here.
The denaturation of the cofactor dependent enzymes from bakers yeast, rabbit muscle and S. pombe by GdnHCl has been examined through loss of catalytic activity and changes in fluorescence and circular dichroism. Only a weak correlation between the denaturation and structural configurations of these enzymes was observed. However, the S. pombe enzyme appeared more resistant to structural perturbation in the presence of its cofactor. This difference was also manifest in the enzyme’s thermostability and proteinase resistance. The sedimentation velocity of this form of the enzyme was increased implying that the observed changes could have resulted from a more compact structure in the presence of the cofactor.
The renaturation of a range of phosphoglycerate mutases has been examined by measuring the regain of activity; structural aspects of this process have been explored using limited proteolysis indicator of bond accessibility.
The bakers yeast enzyme refolds with high efficiency at concentrations between 10 and 50 µg ml-1. Monomeric and dimeric intermediates of refolding exhibiting partial enzymic activity which is sensitive to proteinases have been demonstrated. These partially active species may be similar to a general intermediate formed during the refolding of proteins which has a more “open” structure. Renaturation of the rabbit muscle enzyme is susceptible to the formation of wrong intermolecular aggregates at concentrations above 5µg ml-1. However, refolding intermediates of the rabbit muscle enzyme also have activity which is sensitive to proteinases. The refolding of the S.pomhe enzyme is rapid and concentration independent and resembles the refolding of the subunits of the oligomeric bakers yeast and rabbit muscle enzymes.
The cofactor independent phosphoglycerate mutases from A.nidulans and wheat germ did not exhibit reactivation following denaturation in GdnHCl. This finding may relate to the possible metal ion requirement of these enzymes or reflect an inability of in vitro refolding to mimic accurately in vivo folding in which co-translational processes may act to co-ordinate chain folding and domain pairing.
The findings of the renaturation studies ar considered in relation to the refolding of other proteins which have been examined and to the current model of protein folding in vivo.