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 required for catalytic activity. At which step pyridoxal-P binds in the folding pathway of E. coli serine hydroxymethyltransferase is addressed in this study. E. coli serine hydroxymethyl-transferase is rapidly unfolded to an apparent random coil in 8 M urea. Removal of the urea initiates a complete refolding to the native holoenzyme in less than 10 min at 30 degrees C. Several intermediates on the folding pathway have been identified. The most important information was obtained during folding 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. Continued 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 degrees C bind pyridoxal phosphate. Raising the temperature to 30 degrees C results in the formation of a dimeric enzyme which rapidly binds pyridoxal phosphate forming active enzyme. These studies support the interpretation that pyridoxal phosphate binds only at the end of the folding pathway to dimeric apoenzyme and plays no significant role in the folding mechanism.
Cai
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K
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298, USA.
Schirch
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D
Schirch
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D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D017510:Protein Folding; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D011732:Pyridoxal Phosphate; D012696:Glycine Hydroxymethyltransferase