INHIBITION OF ACROSIN BY SERPINS - A SUICIDE SUBSTRATE MECHANISM

The serpins antithrombin, protease nexin 1, and alpha 1-antitrypsin wi th a reactive-center; arginine (Arg-alpha 1-antitrypsin) were found to inhibit the sperm protease acrosin with varying efficiency. The serpi ns were titrated against acrosin to determine their specific activity with respect to this enzyme. While antithrombin was fully active again st acrosin, more than one molecule of Arg-alpha 1-antitrypsin and prot ease nexin 1 was required to inhibit one molecule of acrosin. In parti cular, only 2.7% of protease nexin 1 molecules interacting with acrosi n formed stable complexes with the enzyme at 37 degrees C and this val ue decreased to 0.03% at 12 degrees C. N-terminal sequence analysis in dicated that acrosin had cleaved protease nexin 1 at its reactive-cent er Arg-Ser bond. The results could be interpreted in terms of protease nexin 1 acting as a suicide substrate for acrosin; after the formatio n of an initial complex, the serpin partitioned between pathways yield ing either inactivated (cleaved) serpin or a stable serpin-enzyme comp lex. The association rate constant (k(ass)) and inhibition constant (K -i) for the stable complexes were determined for each of the serpins b y using slow-binding kinetics. The values of k(ass) were 2 x 10(5), 4 x 10(4), and 5 x 10(3) M(-1) s(-1) for Arg-alpha 1-antitrypsin, antith rombin, and protease nexin 1, respectively. The K-i values for the ser pins were 1 nM or less. Heparin markedly accelerated the inhibition of acrosin by antithrombin and protease nexin 1; at the optimal concentr ation, the degree of heparin acceleration of the inhibition rate was 2 50- and 500-fold for antithrombin and protease nexin 1, respectively. In the presence of heparin, a two-step mechanism could be demonstrated for the acrosin-antithrombin interaction, and the data indicated that the heparin-induced increase in the rate of the acrosin-antithrombin reaction was predominantly due to an increase in the tightness of the initial complex.