REVERSIBLE UNFOLDING OF SHEEP LIVER TETRAMERIC SERINE HYDROXYMETHYLTRANSFERASE

Equilibrium unfolding studies of the tetrameric serine hydroxymethyltr ansferase from sheep liver (SHMT, E.C.2.1.2.1) revealed that the holoe nzyme, apoenzyme and the sodium borohydride-reduced holoenzyme had ran dom coil structures in 8 M urea. In the presence of a non-ionic deterg ent, Brij-35, and polyethylene glycol, the 8 M urea unfolded protein c ould be completely (> 95%) refolded by a 20-fold dilution. The refolde d enzyme was completely active and kinetically similar to the native e nzyme. The midpoint of inactivation of the enzyme occurred at a urea c oncentration that was much below the urea concentration required to br ing about a substantial loss of secondary structure. This observation suggested the occurrence of a 'predenaturation transition' in the unfo lding pathway. The equilibrium urea-induced denaturation curve of holo SHMT showed two transitions. The midpoint of the first transition was 1.2 M, which was comparable to that required for 50% decrease in enzym e activity. Further, 50% release of the pyridoxal-5'-phosphate (PLP) f rom the active site, as monitored by decrease in absorbance at 425 nm, also occurred at about 1.2 M urea. Size exclusion chromatography show ed 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 conce ntration (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 form ation of the active tetramer. The reduced holoenzyme could also be ref olded to its native structure, as observed by fluorescence and CD meas urements, indicating that the presence of covalently linked PLP does n ot affect refolding. The results demonstrate clearly that the dimer is an intermediate in the urea-induced equilibrium unfolding/refolding o f sheep liver SHMT; and PLP, in addition to its role in catalysis, is required for the stabilization of the tetrameric structure of the enzy me. (C) 1998 Elsevier Science B.V.