THE AFFINITY OF PYRIDOXAL 5'-PHOSPHATE FOR FOLDING INTERMEDIATES OF ESCHERICHIA-COLI SERINE HYDROXYMETHYLTRANSFERASE

Escherichia coli serine hydroxymethyltransferase is a 94-kDa homodimer , Each subunit contains a covalently attached pyridoxal-P, which is re quired for catalytic activity, At which step pyridoxal-P binds in the folding pathway of E. coli serine hydroxymethyltransferase is addresse d in this study, E. coli serine hydroxymethyltransferase is rapidly un folded to an apparent random coil in 8 M urea, Removal of the urea ini tiates a complete refolding to the native holoenzyme in less than 10 m in at 30 degrees C. Several intermediates on the folding pathway have been identified, The most important information was obtained during fo lding studies at 4 degrees C. At this temperature, the far-UV circular dichroism spectrum and the fluorescence spectrum of the 3 tryptophan residues become characteristic of the native apoenzyme in less than 10 min, Size exclusion chromatography shows that under these conditions the refolding enzyme is a mixture of monomeric and dimeric species. Co ntinued incubation at 4 degrees C for 60 min results in the formation of only a dimeric species, Neither the monomer nor dimer formed at 4 d egrees C bind pyridoxal phosphate. Raising the temperature to 30 degre es C results in the formation of a dimeric enzyme which rapidly binds pyridoxal phosphate forming active enzyme, These studies support the i nterpretation that pyridoxal phosphate binds only at the end of the fo lding pathway to a dimeric apoenzyme and plays no significant role in the folding mechanism.