Inhibition Mechanism and the Effects of Structure on Activity of Male Reproduction-Related Peptidase Inhibitor Kazal-Type (MRPINK) of Macrobrachium rosenbergii

In our previous reports, a Kazal family serine protease inhibitor, male reproduction-related peptidase inhibitor Kazal-type (MRPINK) has been identified from the prawn, Macrobrachium rosenbergii, and discovered having an inhibitory effect on the sperm gelatinolytic activity. MRPINK was predicated to inhibit chymotrypsin since it contains leucine and proline at P-1 positions of the two domains, respectively. In this report, recombinant MRPINK was as expected found to specifically inhibit chymotrypsin, but no inhibition was detected against trypsin or thrombin. By the analysis of kinetic tests, the inhibition mechanism of MRPINK was determined to be typical competitive model with K-i of 354 nM. To elucidate the effects of structure on activity of MRPINK, the mutants (domain-1 only, domain-2 only, MRPINKP88I, MRPINKL37K, MRPINKL37A, and MRPINKL37G) were prepared and their inhibitory activities assayed. The results showed that domain-2 was the key contributor to the inhibition of chymotrypsin (K-i of 416 nM) and P-1 Pro was crucial for the activity. Nevertheless, whether the P-1 amino acid residue was Leu, or even if it was replaced by Lys, Ala, or Gly, domain-1 was ineffective to the activity.