Reaction of human alpha 2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure.
Human alpha 2-macroglobulin (alpha 2M) half-molecules were prepared by limited reduction and alkylation of the native protein. Reaction with plasmin resulted in nearly quantitative cleavage of the half-molecule Mr approximately 180000 subunits into Mr approximately 90000 fragments. Subunit cleavage was significantly less complete when plasmin was reacted with alpha 2M whole molecules. The plasmin and trypsin binding capacities of the two forms of alpha 2M were compared by using radioiodinated proteases. alpha 2M half-molecules bound an equivalent number of moles of plasmin or trypsin. Native unreduced alpha 2M bound only half as much plasmin as trypsin. These data are consistent with the hypothesis that the two protease binding sites are adjacent in native alpha 2M. alpha 2M half-molecule-plasmin complexes reassociated less readily than half-molecule-trypsin complexes, supporting this interpretation. The frequency of covalent bond formation between plasmin and alpha 2M was considerably higher than that previously observed with other proteases. Approximately 80-90% of the plasmin that reacted with alpha 2M whole molecules or half-molecules became covalently bound. The reactivities of purified alpha 2M-plasmin complexes were compared with small and large substrates. Equivalent kcat/Km values were determined at 22 degrees C for the hydrolysis of H-D-Val-Leu-Lys-p-nitroanilide dihydrochloride by whole molecule-plasmin complex and half-molecule-plasmin complex (40 mM-1 s-1 and 39 mM-1 s-1, respectively, compared with 66 mM-1 s-1 determined for free plasmin).(ABSTRACT TRUNCATED AT 250 WORDS)
D001665:Binding Sites; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism*
D046911:Macromolecular Substances; D000511:alpha-Macroglobulins; D010447:Peptide Hydrolases; D014357:Trypsin; D005341:Fibrinolysin