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

Rose, T., Glaser, P., Surewicz, W. K., Mantsch, H. H., Reinstein, J., Le Blay, K., et al. (1991). Structural and functional consequences of amino acid substitutions in the second conserved loop of Escherichia coli adenylate kinase. The Journal of Biological Chemistry, 266(35), 23654-23659. Retrieved from http://www.jbc.org/cgi/content/abstract/266/35/23654.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0019-AC3A-E Version Permalink: http://hdl.handle.net/21.11116/0000-0002-69BA-7
Genre: Journal Article

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 Creators:
Rose, Thierry, Author
Glaser, Phillipe, Author
Surewicz, Witold K., Author
Mantsch, Henry H., Author
Reinstein, Jochen1, Author              
Le Blay, Karin, Author
Gilles, Anne−Marie, Author
Barzu, Octavian, Author
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1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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 Abstract: 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.

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Language(s): eng - English
 Dates: 1991-07-151991-12-15
 Publication Status: Published in print
 Pages: 6
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
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Title: The Journal of Biological Chemistry
  Other : JBC
Source Genre: Journal
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Publ. Info: Baltimore, etc. : American Society for Biochemistry and Molecular Biology [etc.]
Pages: - Volume / Issue: 266 (35) Sequence Number: - Start / End Page: 23654 - 23659 Identifier: ISSN: 0021-9258
CoNE: https://pure.mpg.de/cone/journals/resource/954925410826_1