The role of pyridoxal phosphate in the refolding of serine hydroxymethyltransferase

Abstract

Escherichia coli serine hydroxymethyltransferase is reversibly unfolded in 1 to 8 M urea solutions in Tris buffer, pH 7.3. Three methods were used to study the refolding process; fluorescence of tryptophan residues, circular dichroism of both secondary structure and pyridoxal phosphate binding, and size separation by high performance liquid chromatography. During unfolding the earliest event is the dissociation of pyridoxal phosphate which occurs in solutions between 1 and 2 M urea. Separation of the dimer subunits occurs between 2.5 and 6 M urea. Complete loss of secondary and tertiary structure occurs above 6.5 M urea. Refolding studies were done at 0.8 M urea in both the presence and absence of pyridoxal phosphate. Greater than 95% of the catalytic activity could be recovered from fully unfolded enzyme in 10 minutes at 30 °C. There was a lag period of 30 seconds before the appearance of active enzyme. The addition of pyridoxal phosphate did not cause a significant change in the refolding kinetics. When refolding was done at 4 °C no active enzyme was formed in a period of several hours, but both secondary and tertiary structure appeared to be like native enzyme. Analysis on high performance liquid chromatography size exclusion gels showed that after a few minutes of refolding at 4 °C the enzyme was a mixture of monomer and dimer species. After one hour the enzyme was all dimer. Analysis by circular dichroism and Trp fluorescence during this period showed that pyridoxal phosphate did not bind to either the monomer or dimer formed at 4 °C. We conclude that pyridoxal phosphate binds only to the native apoenzyme and does not play a role in the refolding process of this enzyme.