Inhibition of proteases by alpha 2-macroglobulin. The role of lysyl amino groups of trypsin in covalent complex formation.

Abstract

The lysyl amino groups of bovine trypsin were covalently modified by acetylation, succinylation, or reductive methylation. The enzymatically active derivatives were still capable of reaction with alpha 2-macroglobulin (alpha 2M), although to a lesser extent than native enzyme. The resulting enzyme-alpha 2M complexes, however, were much more susceptible to dissociation by sodium dodecyl sulfate than complexes formed with unmodified trypsin. The bound modified enzymes could be released from the alpha 2M complex with an excess of native thrombin. In addition, anhydrotrypsin displaced methyl trypsin from its complex and the anhydro derivative was bound in its place. The data provide evidence for two types of noncovalent intermediates; those formed from lysyl-modified enzymes show proteolysis of the alpha 2M to the nominal 85,000 fragment, whereas anhydrotrypsin forms a complex with apparently intact alpha 2M chains. A model is proposed for the reaction of alpha 2M with proteases in which one or both of these noncovalent intermediates is formed. Conversion of this form(s) to a stable covalent complex requires unmodified lysyl amino groups on the enzyme, suggesting that these groups may form a covalent bond with the inhibitor, possibly at the site at which methylamine binds.