Escherichia coli ribonuclease HI, which requires divalent cations (Mg2+ or Mn2+) for activity, was thermostabilized by 2.6-3.0 kcal/mol in the presence of the Mg2+, Mn2+, or Ca2+ ion, probably because the negative charge repulsion around the active site was canceled upon the binding of these metal ions. The dissociation constants were determined to be 0.71 mM for Mg2+, 0.035 mM for Mn2+, and 0.16 mM for Ca2+. Likewise, various active site mutants at Asp10, Glu48, Asp70, or Asp134 were thermostabilized by 0.4-3.0 kcal/mol in the presence of the Mg2+ ion, suggesting that this ion binds to these mutant proteins as well. The dissociation constants of Mg2+ were determined to be 9.8 mM for D10N, 1.1 mM for E48Q, 18.8 mM for D70N, and 1.8 mM for D134N. Thus, the mutation of Asp10 or Asp70 to Asn considerably impairs the Mg2+ binding, whereas the mutation of Glu48 to Gln or Asp134 to Asn does not. Comparison of the thermal stability of the mutant proteins with that of the wild-type protein in the absence of the Mg2+ ion suggests that the negative charge repulsion between Asp10 and Asp70 is responsible for the binding of the metal cofactor. Glu48 may be required to anchor a water molecule, which functions as a general acid. Study holds ProTherm entries: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 13216, 13217, 13218, 13219, 13220, 13221, 13222, 13223, 13224, 13225, 13226, 13227, 13228, 13229, 13230, 13231, 13232, 13233, 13234, 13235, 13236, 13237, 13238, 13239, 13240, 13241 Extra Details: Escherichia coli ribonuclease HI; thermal stability; catalytic role;,dissociation constant
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:14 p.m.
|Number of data points||97|
|Proteins||Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI|
|Assays/Quantities/Protocols||Experimental Assay: ddG temp:50.0 C, pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: ddG buffers:glycine-NaOH: 50 mM, temp:47.0 C, pH:9.0 ; Experimental Assay: Tm pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: dHvH ; Experimental Assay: activity ; Experimental Assay: Tm pH:9.0, buffers:glycine-NaOH: 50 mM ; Derived Quantity: dTm pH:3.0, buffers:glycine-HCl: 10 mM ; Derived Quantity: dTm pH:9.0, buffers:glycine-NaOH: 50 mM|
|Libraries||Mutations for sequence MLKQVEIFTDGSCLGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV|