Mass spectrometric determination of association constants of adenylate kinase with two noncovalent inhibitors.
Noncovalent complexes between chicken muscle adenylate kinase and two inhibitors, P(1),P(4)-di(adenosine-5')tetraphosphate (Ap4A) and P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A), were investigated with electrospray ionization mass spectrometry under non-denaturing conditions. The nonconvalent nature and the specificity of the complexes are demonstrated with a number of control experiments. Titration experiments allowed the association constants for inhibitor binding to be determined. Problems with concentration dependent ion yields are circumvented by a data evaluation method that is insensitive to the overall ionization efficiency. The K(a) values found were 9.0 x 10(4) M(-1) (Ap4A) and 4.0 x 10(7) M(-1) (Ap5A), respectively, in very good agreement with available literature data.
Daniel
Jürg M
JM
Department of Chemistry, Swiss Federal Institute of Technology, ETH, CH-8093, Zürich, Switzerland.
McCombie
Gregor
G
Wendt
Silke
S
Zenobi
Renato
R
D000263:Adenylate Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D000818:Animals; D002645:Chickens; D004791:Enzyme Inhibitors / Q000737:chemistry*; D015394:Molecular Structure; D018482:Muscle, Skeletal / Q000201:enzymology; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization
D004791:Enzyme Inhibitors; D000263:Adenylate Kinase