Spermine stabilization of folded ribonuclease T sub(1)

• Published in: The Journal of biological chemistry Vol. 265; no. 13; pp. 7127 - 7137
• Main Authors: Walz, FG Jr, Kitareewan, S
• Format: Journal Article
• Language: English
• Published: 01-01-1990
• Subjects: spermine; thermal stability
• ISSN: 0021-9258

The interaction of ribonuclease T sub(1) with tetraprotonated spermine (SPM super(4+)), Mg super(2+), phosphate and other ionic ligands at pH 6.0 was investigated in binding experiments at 25 degree C and/or by their effects on the midpoint temperature for thermal unfolding of the enzyme. SPM super(4+) binding with the native protein at 25 degree C was characterized by an association constant of similar to 2 x 10 super(4) M super(-1). This ligand also binds to the unfolded protein but with a similar to 35-fold lower affinity. Phosphate binds at the active site whereas Mg super(2+) and SPM super(4+) 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 SPM super(4+) binding with the enzyme does not affect its catalytic activity.