Study of noncovalent enzyme—inhibitor complexes and metal binding stoichiometry of matrilysin by electrospray ionization mass spectrometry

摘要: Electrospray ionization mass spectrometry (ESI-MS) was used to study the noncovalent metallo-enzyme—inhibitor complexes of matrilysin (a matrix metalloproteinase 18,720 u) under gentle experimental conditions and determine metal ion association stoichiometries in both free enzyme complexes. The were found be highly sensitive solution pH changes. At 2.2 existed as metal-free apo-matrilysin not capable binding an inhibitor. 4.5–7.0 associated specifically with zinc calcium cations became active inhibitor binding. Although cofactors varied (zero two and/or ions) dependent on pH, predominant form enzyme—inhibitor range 4.5–7.0, contrast, always had stoichiometry 2Zn + 2Ca, which same most metallo-enzyme at optimal 7. activity onset 4.5 mostly apo-enzyme (but a conformation different from denatured one 2.2) bound slowly (time constant ∼ 2.5 min) complex. Of inhibitors studied, higher also produced larger signals for complex solvent-free gas phase, pointed feasibility use ESI-MS screening studies.