Guanidine hydrochloride-induced reversible unfolding of sheep liver serinehydroxymethyltransferase

Equilibrium unfolding studies of sheep liver tetrameric serine hydroxymethy ltransferase (SHMT, EC 2.1.2.1) revealed that the enzyme assumed apparent r andom coil structure above 3 M guanidine hydrochloride (GdnHCl). In the pre sence of non-ionic detergent Brij-35 and polyethylene glycol, the 6 M GdnHC l unfolded enzyme could be completely (>95%) refolded by a 40-fold dilution . The refolded enzyme was fully active and had kinetic constants similar to the native enzyme. The midpoint of inactivation (0.12 M GdnHCl) was well b elow the midpoint of unfolding (1.6+/-0.1 M GdnHCl) as monitored by far UV CD at 222 nm. In the presence of PLP, the midpoint of inactivation shifted to a higher concentration of GdnHCl (0.6 M) showing that PLP stabilizes the quaternary structure of the enzyme. However, 50% release of pyridoxal-5'-p hosphate (PLP) from the active site occurred at a concentration (0.6 M) hig her than the midpoint of inactivation suggesting that GdnHCl may also act a s a competitive inhibitor of the enzyme at low concentrations which was con firmed by activity measurements. PLP was not required for the initiation of refolding and inactive tetramers were the end products of refolding which could be converted to active tetramers upon the addition of PLP. Size exclu sion chromatography of the apoenzyme showed that the tetramer unfolds via t he intermediate formation of dimers. Low concentrations (0.3-0.6 M) of GdnH Cl stabilized at least one intermediate which was in slow equilibrium with the dimer. The binding of ANS was maximum at 0.4-0.6 M GdnHCl suggesting th at the unfolding intermediate that accumulates at this concentration is les s compact than the native enzyme.