Structural and functional consequences of amino acid substitutions in the second conserved loop of Escherichia coli adenylate kinase

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TypeArticle
Journal titleThe Journal of Biological Chemistry
ISSN1083-351X
Volume266
Issue35
Pages2365423659
AbstractAll 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.
Publication date
PublisherThe American Society for Biochemistry and Molecular Biology, Inc.
Linkhttp://www.jbc.org/content/266/35/23654.abstract
LanguageEnglish
AffiliationNational Research Council Canada
Peer reviewedYes
NPARC number23001317
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Record identifiere1ec5226-e1ea-40c7-b817-70c40dc13eff
Record created2017-01-17
Record modified2017-01-17
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