Abstract

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. Study holds ProTherm entries: 5173 Extra Details: ionic ligands; association constant; polyanionic locus;,alpha-helix; catalytic properties

Submission Details

ID: iBzycwwB

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

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