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. (C) 2003 American Society for Mass Spectrometry.