Human glycine N-methyltransferase is unfolded by urea through a compact monomer state

Abstract

Human recombinant glycine N-methyltransferase (GNMT) unfolding by urea was studied by enzyme activity, size-exclusion chromatography, fluorescence spectroscopy, and circular dichroism. Urea unfolding of GNMT is a two-step process. The first transition is a reversible dissociation of the GNMT tetramer to compact monomers in 1.0–3.5 M urea with enzyme inactivation. The compact monomers were characterized by Stokes radius ( R s) of 40.7 Å equal to that of globular proteins with the same molecular mass as GNMT monomers, absence of exposure of tryptophan residues into solvent, and presence of about 50% of secondary structure of native protein. The second step of GNMT unfolding is a reversible transition of monomers from compact to a fully unfolded state with R s of 50 Å, exposed tryptophan residues, and disrupted secondary structure in 8 M urea.