Inhibition Mechanism and the Effects of Structure on Activity of Male Reproduction-Related Peptidase Inhibitor Kazal-Type (MRPINK) of Macrobrachium rosenbergii |
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Authors: | Ye Li Ye-Qing Qian Wen-Ming Ma Wei-Jun Yang |
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Affiliation: | (1) Institute of Cell Biology and Genetics, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058, People’s Republic of China;(2) State Conservation Center for Gene Resources of Wildlife and the Key Laboratory of Conservation Genetics and Reproductive Biology for Wild Animals of the Ministry of Education, Hangzhou, Zhejiang, 310058, People’s Republic of China |
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Abstract: | 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 P1 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 P1 Pro was crucial for the activity. Nevertheless, whether the P1 amino acid residue was Leu, or even if it was replaced by Lys, Ala, or Gly, domain-1 was ineffective to the activity. |
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Keywords: | Kazal-type peptidase inhibitor Recombinant Mutant Domain P1 position |
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