Abstract
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.
Highlights
(EC 2.1.2.1) is a 94-kDa homodimer that catalyzes the interconversion of serine and glycine with H4PteGlun serving as the one-carbon carrier
Formation of the external aldimine by saturation with serine causes a significant increase of 8 °C in the Tm of the protein and, with other evidence, suggests that the eSHMT1⁄7serine complex is in the more stable closed form (18)
Because there was no fluorescent probe in the section between residues 225 and 276, the only available evidence as to its structure in M and DЈ was its sensitivity to digestion with subtilisin. This segment of the amino acid sequence is important because it contains the active site Lys[229], which binds PLP, but several other residues that have been shown to be involved in catalysis (20, 21)
Summary
Materials—t-Butoxycarbonyl-Lys was purchased from Advanced Chemtech (Louisville, KY). Equilibrium unfolding-refolding was performed by adding a concentrated solution of either wild-type or mutant PyP-eSHMTs to a series of urea concentrations in the Tris buffer (4). These solutions were incubated for 5 h at 30 °C before analysis by fluorescence spectrometry. The fluorescence equilibrium unfolding-refolding data were converted to the apparent fraction of native protein (Fapp) and plotted versus urea concentration, as defined by Equation 4. Kinetic Refolding Properties of PyP-eSHMT—Unfolding of eSHMT was performed by diluting 5-fold a concentrated enzyme solution (7.5 mg/ml) into 10 M urea. Native controls were made in 0.8 M urea with Tris buffer, and the final protein concentration was exactly the same as the samples used in these refolding experiments. Samples were loaded and electrophoresis was performed at 12 mA for 1 h
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