CHARACTERIZATION OF METAL AND NUCLEOTIDE LIGANDED FORMS OF ADENYLATE KINASE BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

Complexes of adenylate kinase from Escherichia coli, Bacillus subtilis , and Bacillus stearothermophilus with the bisubstrate nucleotide anal og P-1,P-5-di(adenosine 5')-pentaphosphate and with metal ions (Zn2+ a nd/or Mg2-) were analyzed by electrospray ionization mass spectrometry . P-1,P-5-di(adenosine 5')pentaphosphate adenylate kinase complex was detected in the positive mode at pH as low as 3.8. Binding of nucleoti de to adenylate kinase stabilizes the overall structure of the protein and preserves the Zn2+ chelated form of the enzyme from the gram-posi tive organisms, In this way, it is possible in a single mass spectrome try experiment to screen metal-chelating adenylate kinases, without us e of radioactively labeled compounds, Binding of Mg2+ to enzyme via P- 1,P-5-di(adenosine 5')-pentaphosphate was also demonstrated by mass sp ectrometry. Although no amino acid side chain in adenylate kinase is s upposed to interact with Mg2+, Asp(93) in porcine muscle cytosolic enz yme, equivalent to Asp(84) in the E. coli adenylate kinase, was propos ed to stabilize the nucleotide . Mg2+ complex via water molecules. (C) 1997 Academic Press.