Bacillus subtilis neutral protease : III. Exchange properties of zinc and preparation of some metal derivatives

Abstract

The Bacillus subtilis neutral protease is inhibited by an excess of Hg ++, Cd ++, Pb ++, and Ni ++. Co ++, Mn ++, and Fe ++ do not inhibit in the same concentration range. The zinc in the native enzyme can be replaced by Hg ++, Cd ++, Pb ++, and 65Zn ++. Conditions for preparing highly purified preparations of the first four metal derivatives are described. There is a direct replacement of one mole of metal for one mole of zinc except for the Hg-derivatives, which bind 2 moles of metal. These derivatives hydrolyzed hippuryl- l-leucinamide at about the same rate, except possibly for the Pb and Cd derivatives, but hydrolyzed casein at different rates. These experiments do not elucidate the role of the metal ions but indicate that a variety of metal ions can replace the zinc in the native enzyme. The metal binding site appears to be different from that reported for carbonic anhydrase or carboxypeptidase, since the Cu derivative is colorless in the case of neutral protease.