Spermine stabilization of folded ribonuclease T1.
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.
Walz
F G
FG
Department of Chemistry, Kent State University, Ohio 44242.
Kitareewan
S
S
D001224:Aspartic Acid; D004722:Endoribonucleases / Q000378:metabolism*; D004795:Enzyme Stability; D005971:Glutamates; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013096:Spermine / Q000378:metabolism / Q000494:pharmacology*; D013816:Thermodynamics
D005971:Glutamates; D008024:Ligands; D013096:Spermine; D001224:Aspartic Acid; D018698:Glutamic Acid; D004722:Endoribonucleases; D006163:Ribonuclease T1; D008274:Magnesium