FOCUS: NONCOVALENT INTERACTIONS Mass Spectrometric Determination of Association Constants of Adenylate Kinase with Two Noncovalent Inhibitors

Noncovalent complexes between chicken muscle adenylate kinase and two inhibitors, P1,P4-di(adenosine-5')tetraphosphate (Ap4A) and P1,P5-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 Ka values found were 9.0 10 4 M1 (Ap4A) and 4.0 107 M1 (Ap5A), respectively, in very good agreement with available literature data. (J Am Soc Mass Spectro