STUDY OF NONCOVALENT ENZYME-INHIBITOR COMPLEXES AND METAL-BINDING STOICHIOMETRY OF MATRILYSIN BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

Electrospray ionization mass spectrometry (ESI-MS) was used to study t he noncovalent metallo-enzyme-inhibitor complexes of matrilysin (a mat rix metalloproteinase of mass 18,720 u) under gentle experimental cond itions and to determine the metal ion association stoichiometries in b oth the free enzyme and the complexes. The metal association stoichiom etries of the free matrilysin were found to be highly sensitive to sol ution pH changes. At pH 2.2 the enzyme existed as metal-free apo-matri lysin and was not capable of binding an inhibitor. At pH 4.5-7.0 the e nzyme associated specifically with zinc and calcium cations and became active in inhibitor binding. Although the stoichiometries of the meta l cofactors varied (zero to two zinc and/or calcium ions) in the free enzyme dependent on solution pH, the predominant form of the enzyme-in hibitor complexes in the pH range of 4.5-7.0, in contrast, always had the metal association stoichiometry of 2Zn + 2Ca, which was the same s toichiometry the most active free metallo-enzyme had at the optimal pH of 7. At the activity onset FH of 4.5 matrilysin existed mostly as ap o-enzyme (but in a conformation different from the denatured one at pH 2.2) and bound to an inhibitor slowly (time constant similar to 2.5 m in) to form the noncovalent metallo-enzyme-inhibitor complex. Of the t wo inhibitors studied, the one with the higher solution binding consta nt also produced larger ion signals for the noncovalent complex in the solvent-free gas phase, which pointed to the feasibility of the use o f ESI-MS for inhibitor screening studies.