Towards a mechanism of AMP‐substrate inhibition in adenylate kinase from Escherichia coli

Crystallographic studies on adenylate kinase (AK) suggest that binding of ATP causes the LID domain of the enzyme to close over the ATP molecule (Schlauderer et al. (1996) J. Mol. Biol. 256, 223–227). The method of time‐resolved fluorescence resonance energy transfer was applied to study the proposed structural change in AK from Escherichia coli. Two active derivatives of the (C77S, A73C, V142C)‐AK mutant containing the excitation energy donor attached to one of the two cysteine residues and the acceptor attached to the other cysteine were prepared to monitor displacements of the LID domain in response to substrate binding. Binding of either ATP or AMP was accompanied by a ∼ 9 A decrease in the interprobe distances suggesting LID domain closure. Closure of the LID domain in response to AMP binding may be a possible reason for the strong AMP‐substrate inhibition known for E. coli AK.

[1]  M Gerstein,et al.  Domain closure in adenylate kinase. Joints on either side of two helices close like neighboring fingers. , 1993, Journal of molecular biology.

[2]  G. Schulz,et al.  Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases. , 1995, Structure.

[3]  R L Blakeley,et al.  Ellman's reagent: 5,5'-dithiobis(2-nitrobenzoic acid)--a reexamination. , 1979, Analytical biochemistry.

[4]  L. Noda.  8 Adenylate Kinase , 1973 .

[5]  G. Schulz,et al.  Structure of a mutant adenylate kinase ligated with an ATP-analogue showing domain closure over ATP. , 1996, Journal of molecular biology.

[6]  P. Marlière,et al.  Conservative replacement of methionine by norleucine in Escherichia coli adenylate kinase. , 1988, The Journal of biological chemistry.

[7]  G. Phillips,et al.  Assignment of the nucleotide binding sites and the mechanism of substrate inhibition of Escherichia coli adenylate kinase , 1991, Proteins.

[8]  G. Schulz,et al.  Adenylate kinase motions during catalysis: an energetic counterweight balancing substrate binding. , 1996, Structure.

[9]  W. Mantulin,et al.  Substrate binding causes movement in the ATP binding domain of Escherichia coli adenylate kinase. , 1996, Biochemistry.

[10]  G. Schulz,et al.  Structure of the complex between adenylate kinase from Escherichia coli and the inhibitor Ap5A refined at 1.9 A resolution. A model for a catalytic transition state. , 1992, Journal of molecular biology.

[11]  E. Haas,et al.  Nonlocal interactions stabilize compact folding intermediates in reduced unfolded bovine pancreatic trypsin inhibitor. , 1992, Biochemistry.

[12]  I R Vetter,et al.  Fluorescence and NMR investigations on the ligand binding properties of adenylate kinases. , 1990, Biochemistry.

[13]  C. Cantor,et al.  The use of singlet-singlet energy transfer to study macromolecular assemblies. , 1978, Methods in enzymology.

[14]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[15]  A. Danchin,et al.  Structural and catalytic characteristics of Escherichia coli adenylate kinase. , 1987, The Journal of biological chemistry.

[16]  G. Phillips,et al.  The closed conformation of a highly flexible protein: The structure of E. coli adenylate kinase with bound AMP and AMPPNP , 1994, Proteins.

[17]  K. Diederichs,et al.  The refined structure of the complex between adenylate kinase from beef heart mitochondrial matrix and its substrate AMP at 1.85 A resolution. , 1991, Journal of molecular biology.

[18]  W. Melhuish.  A STANDARD FLUORESCENCE SPECTRUM FOR CALIBRATING SPECTRO-FLUOROPHOTOMETERS , 1960 .

[19]  E. Haas,et al.  Domain closure in adenylate kinase. , 1996, Biochemistry.

[20]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[21]  G. Schulz,et al.  High‐resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer , 1995, Protein science : a publication of the Protein Society.

[22]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.