All known nucleoside monophosphate kinases contain an invariant sequence Asp-Gly-Phe(Tyr)-Pro-Arg. In order to understand better the structural and functional role of individual amino acid residues belonging to the above sequence, three mutants of Escherichia coli adenylate kinase (D84H, G85V, and F86L) were produced by site-directed mutagenesis. Circular dichroism spectra revealed that the secondary structure dichroism spectra revealed that the secondary structure of all three mutant proteins is very similar to that of the wild-type enzyme. However, each of the substitutions resulted in a decreased thermodynamic stability of the protein, as indicated by differential scanning calorimetry measurements and equilibrium unfolding experiments in guanidine HCl. The destabilizing effect was most pronounced for the G85V mutant, in which case the denaturation temperature was decreased by as much as 11 degrees C. The catalytic activity of the three mutants represented less than 1% of that of the wild-type enzyme. Furthermore, for the D84H-modified form of adenylate kinase, the impaired binding of nucleotide substrates was accompanied by a markedly decreased affinity for magnesium ion. These observations support the notion that Asp84 is directly involved in binding of nucleotide substrates and that this binding is mediated by interaction of the aspartic acid residue with divalent cation. The two remaining residues probed in this study, Gly85 and Phe86, belong to a beta-turn which appears to play a major role in stabilizing the three-dimensional structure of adenylate kinase. Study holds ProTherm entries: 98, 99, 100, 101, 13184, 13185, 13186 Extra Details: Escherichia coli adenylate kinase; thermodynamic stability;,differential scanning calorimetry; three-dimensional structure
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:14 p.m.
|Number of data points||18|
|Proteins||Adenylate kinase ; Adenylate kinase|
|Assays/Quantities/Protocols||Experimental Assay: ddG ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: activity_kd ; Derived Quantity: dTm|
|Libraries||Mutations for sequence MRIILLGAPGAGKGTQAQFIMEKYGIPQISTGDMLRAAVKSGSELGKQAKDIMDAGKLVTDELVIALVKERIAQEDCRNGFLLDGFPRTIPQADAMKEAGINVDYVLEFDVPDELIVDRIVGRRVHAPSGRVYHVKFNPPKVEGKDDVTGEELTTRKDDQEETVRKRLVEYHQMTAPLIGYYSKEAEAGNTKYAKVDGTKPVAEVRADLEKILG|