Mechanism of .alpha.2-macroglobulin-proteinase interactions. Studies with trypsin and plasmin

Abstract

The time course of the interaction of [human] .alpha.2-macroglobulin with trypsin and with plasmin was studied by measuring the generation of thiol groups, the concentration of .alpha.2-macroglobulin subunits cleaved at the bait regions and the change in intrinsic protein fluorescence of .alpha.2-macroglobulin-enzyme reaction mixtures as functions of time. The interaction of .alpha.2-macroglobulin with trypsin was very fast but could be studied in the presence of benzamidine, a rather strong competitive inhibitor of trypsin. .alpha.2-Macroglobulin-proteinase reactions, known to involve specific limited proteolysis of the bait regions, gross conformational changes and cleavage of the internal .beta.-cysteinyl-.gamma.-glutamyl thiol esters of native .alpha.2-macroglobulin, may proceed via at least 2 different reaction pathways determined by the nature of or the concentration of the reacting proteinase. After initial cleavage of one bait region at high proteinase activity the next step presumably is a fast cleavage of a second bait region before any substantial rearrangements leading to generation of thiol groups and the final incorporation of the proteinase occur. At low proteinase activity no further bait region cleavages occur and only the 2 thiol groups of half of the .alpha.2-macroglobulin molecule are generated in the final 1:1 complex. Estimates of the initial association rate constants of the .alpha.2-macroglobulin-trypsin and the .alpha.2-macroglobulin-plasmin reactions were 2 .times. 107 M-1 s-1 and 5 .times. 105 M-1 s-1, respectively, apparently corresponding to the overall rate constants of the cleavage of one bait region in native .alpha.2-macroglobulin, that is, kc/Km of that enzymatic step. After rearrangements within the complexes with generation of 2 of the possible 4 thiol groups, a further bait region cleavage step was more slow; the apparent rate constants were 1.3 .times. 106 M-1 s-1 for trypsin and 3 .times. 103 M-1 s-1 for plasmin.