J. Biol. Chem., Vol. 265, Issue 13, 7127-7137, 05, 1990
Spermine stabilization of folded ribonuclease T1
FG Walz Jr and S Kitareewan
Department of Chemistry, Kent State University, Ohio 44242.
The interaction of ribonuclease T1 with tetraprotonated spermine (SPM4+),
Mg2+, phosphate and other ionic ligands at pH 6.0 was investigated in
binding experiments at 25 degrees C and/or by their effects on the midpoint
temperature for thermal unfolding of the enzyme. SPM4+ binding with the
native protein at 25 degrees C was characterized by an association constant
of approximately 2 x 10(4) M- 1. This ligand also binds to the unfolded
protein but with a approximately 35-fold lower affinity. Phosphate binds at
the active site whereas Mg2+ and SPM4+ cations compete for binding at a
polyanionic locus that probably involves residues Glu-28, Asp-29, and
Glu-31 at the C-terminal end of the alpha-helix. Steady-state kinetic
studies using minimal RNA substrates demonstrated that SPM4+ binding with
the enzyme does not affect its catalytic activity. SPM4+ also
preferentially binds with the folded form of the disulfide-reduced enzyme
which has the same or slightly enhanced catalytic properties compared with
native ribonuclease T1. The unfolding rate for the native protein in 8 M
urea was approximately 8-fold lower in the presence of 0.05 M SPM4+. SPM4+
appears to increase the amplitude of an unobserved fast phase(s) for
refolding of the native enzyme. A single kinetic phase characterized
refolding of the reduced enzyme which was slightly faster than the slowest
refolding phase for the native form.
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