Structural studies on folding intermediates of serine hydroxymethyltransferase using fluorescence resonance energy transfer.

Previous studies have demonstrated that the in vitro folding pathway of Escherichia coli serine hydroxymethyltransferase has both monomer and dimer intermediates that are stable for periods of minutes to hours at 4 degrees C (Cai K., Schirch, D., and Schirch, V. (1995) J. Biol. Chem. 270, 19294-19299). Single Trp mutant enzymes were constructed and used in combination with other methods to show that on the folding pathway of this enzyme two domains rapidly fold to form a monomer in which the amino-terminal 55 amino acid residues and a segment around the active site region of Lys229 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate-determining conformational change in which the unstructured segments assume their native state (Cai, K. , and Schirch, V. (1996) J. Biol. Chem. 271, 2987-2994). To further assess the kinetics and structural details of the intermediates during folding, fluorescence energy transfer and fluorescence anisotropy measurements were made of the three Trp residues and pyridoxal 5'-phosphate, attached covalently to the active site by reduction to a secondary amine by sodium cyanoborohydride. These studies confirmed that the basic kinetic folding pathway remained the same in the reduced enzyme as compared to the earlier studies with the apoenzyme. Both equilibrium and kinetic intermediates were identified and their structural characteristics determined. The results show that the active site Lys229-bound pyridoxyl 5'-phosphate remains more than 50 angstroms from any Trp residues until the final rate-determining conformational change when it approaches each Trp residue at the same rate. The environment of each Trp residue and the pyridoxyl phosphate in both an equilibrium folding intermediate and a kinetic folding intermediate are described.