Presence versus absence of hydrogen bond donor Tyr‐39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors

Moh'd A. Salameh; Alexei S. Soares; Alexandre Alloy; Evette S. Radisky

doi:10.1002/pro.2097

journal article Jun 25, 2012

Presence versus absence of hydrogen bond donor Tyr‐39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors

Moh'd A. Salameh Alexei S. Soares Alexandre Alloy Evette S. Radisky

Protein Science Vol. 21 No. 8 pp. 1103-1112 · Wiley

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Abstract

AbstractMesotrypsin displays unusual resistance to inhibition by polypeptide trypsin inhibitors and cleaves some such inhibitors as substrates, despite a high degree of conservation with other mammalian trypsins. Substitution of Arg for the generally conserved Gly‐193 has been implicated as a critical determinant of the unusual behavior of mesotrypsin toward protein protease inhibitors. Another relatively conserved residue near the trypsin active site, Tyr‐39, is substituted by Ser‐39 in mesotrypsin. Tyr‐39, but not Ser‐39, forms a hydrogen bond with the main chain amide nitrogen of the P4′ residue of a bound protease inhibitor. To investigate the role of the Tyr‐39 H‐bond in trypsin‐inhibitor interactions, we reciprocally mutated position 39 in mesotrypsin and human cationic trypsin to Tyr‐39 and Ser‐39, respectively. We assessed inhibition constants and cleavage rates of canonical protease inhibitors bovine pancreatic trypsin inhibitor (BPTI) and the amyloid precursor protein Kunitz protease inhibitor domain by mesotrypsin and cationic trypsin variants, finding that the presence of Ser‐39 relative to Tyr‐39 results in a 4‐ to 13‐fold poorer binding affinity and a 2‐ to 18‐fold increase in cleavage rate. We also report the crystal structure of the mesotrypsin‐S39Y•BPTI complex, in which we observe an H‐bond between Tyr‐39 OH and BPTI Ile‐19 N. Our results indicate that the presence of Ser‐39 in mesotrypsin, and corresponding absence of a single H‐bond to the inhibitor backbone, makes a small but significant functional contribution to the resistance of mesotrypsin to inhibition and the ability of mesotrypsin to proteolyze inhibitors.

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Details

Published: Jun 25, 2012
Vol/Issue: 21(8)
Pages: 1103-1112
License: View

Authors

Funding

Florida Department of Health Award: 07BN-07

US National Cancer Institute Award: CA091956

US Department of Defense Award: PC094054

Cite This Article

Moh'd A. Salameh, Alexei S. Soares, Alexandre Alloy, et al. (2012). Presence versus absence of hydrogen bond donor Tyr‐39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors. Protein Science, 21(8), 1103-1112. https://doi.org/10.1002/pro.2097

Presence versus absence of hydrogen bond donor Tyr‐39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors

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