Venkatesha, B and Udgaonkar, Jayant B and Rao, N Appaji and Savithri, HS (1998) Reversible unfolding of sheep liver tetrameric serine hydroxymethyltransferase. In: Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1384 (01). pp. 141-152.
Reversible_unfolding_of_sheep.pdf - Published Version
Restricted to Registered users only
Download (252Kb) | Request a copy
Equilibrium unfolding studies of the tetrameric serine hydroxymethyltransferase from sheep liver (SHMT, E.C.184.108.40.206) revealed that the holoenzyme, apoenzyme and the sodium borohydride-reduced holoenzyme had random coil structures in 8 M urea. In the presence of a non-ionic detergent, Brij-35, and polyethylene glycol, the 8 M urea unfolded protein could be completely (> 95%) refolded by a 20-fold dilution. The refolded enzyme was completely active and kinetically similar to the native enzyme. The midpoint of inactivation of the enzyme occurred at a urea concentration that was much below the urea concentration required to bring about a substantial loss of secondary structure. This observation suggested the occurrence of a 'predenaturation transition' in the unfolding pathway. The equilibrium urea-induced denaturation curve of holoSHMT showed two transitions. The midpoint of the first transition was 1.2 M, which was comparable to that required for 50% decrease in enzyme activity. Further, 50% release of the pyridoxal-5'-phosphate (PLP) from the active site, as monitored by decrease in absorbance at 425 nm, also occurred at about 1.2 M urea. Size exclusion chromatography showed that the tetrameric SHMT unfolds via the intermediate formation of dimers. This dissociation occurred at a much lower urea concentration (0.15 M) in the unfolding of the apoenzyme, and at a higher urea concentration (1.2 M) in the unfolding of holoenzyme, thereby demonstrating the involvement of PLP in stabilizing the quaternary structure of the enzyme. Size exclusion chromatography of the refolding intermediates demonstrated that the cofactor shifts the equilibrium towards the formation of the active tetramer. The reduced holoenzyme could also be refolded to its native structure, as observed by fluorescence and CD measurements, indicating that the presence of covalently linked PLP does not affect refolding. The results demonstrate clearly that the dimer is an intermediate in the urea-induced equilibrium unfolding/refolding of sheep liver SHMT; and PLP, in addition to its role in catalysis, is required for the stabilization of the tetrameric structure of the enzyme.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belongs to Elsevier Science.|
|Keywords:||urea denaturation;reversible unfolding;oligomeric protein; PLP-dependent enzyme;SHMT.|
|Department/Centre:||Division of Biological Sciences > Biochemistry|
|Date Deposited:||24 Dec 2009 09:25|
|Last Modified:||19 Sep 2010 05:26|
Actions (login required)