Changes in MM-CK conformational mobility upon formation of the ADP-Mg(2+)-NO(3)(-)-creatine transition state analogue complex as detected by hydrogen/deuterium exchange.
In the presence of ADP, Mg(2+), creatine, and the planar nitrate ion, creatine kinase isoenzymes undergo significant structural changes accompanying the formation of a very stable transition state analogue complex (TSAC). We have compared, by using hydrogen/deuterium exchange followed by proteolysis of the labeled enzyme and mass spectrometric analysis of the peptic peptides, the backbone dynamics fluctuations of the free enzyme and those of the TSAC. In most peptides, exchange is not affected by ligand binding, except that observed in seven areas located in or at the entrance to the active site, where some protection is detected. On the basis of a comparison with the three-dimensional structures of free or liganded guanidino kinases, four of these peptides (residues 54-72, 226-234, 287-311, and 315-333) can be considered part of the substrate binding site. The other three (residues 162-186, 193-201, and 202-224) are not directly involved in the binding of substrates and are located in a dynamic domain, which allows the enzyme to properly align the substrates for optimal catalysis.
Mazon
Hortense
H
UMR CNRS 5013, Biomembranes et enzymes associés, Université Claude Bernard Lyon 1, 43, boulevard du 11 Novembre 1918, 69622 Villeurbanne cedex, France.
Marcillat
Olivier
O
Forest
Eric
E
Vial
Christian
C
D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003401:Creatine / Q000737:chemistry* / Q000378:metabolism; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D006859:Hydrogen / Q000737:chemistry; D007527:Isoenzymes; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009566:Nitrates / Q000737:chemistry* / Q000378:metabolism; D010434:Pepsin A / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011487:Protein Conformation; D011817:Rabbits; D021241:Spectrometry, Mass, Electrospray Ionization
D007527:Isoenzymes; D009566:Nitrates; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D006859:Hydrogen; D003402:Creatine Kinase; D010434:Pepsin A; D008274:Magnesium; D003401:Creatine