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Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys 14 Cys 39 bond into the ovomucoid third domain from silver pheasant
Author(s) -
Hemmi Hikaru,
Kumazaki Takashi,
Kojima Shuichi,
Yoshida Takuya,
Ohkubo Tadayasu,
Yokosawa Hideyoshi,
Miura Kinichiro,
Kobayashi Yuji
Publication year - 2011
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.1381
Subject(s) - chemistry , active site , hydrolysis , stereochemistry , peptide bond , reaction rate constant , streptomyces griseus , peptide , enzyme , biochemistry , kinetics , streptomyces , biology , bacteria , genetics , physics , quantum mechanics
P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys 14 Cys 39 bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys 14 Cys 39 bond into the flexible N‐terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH‐independent hydrolysis constant ( K hyd °) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K hyd °, but has no effect on the thermal stability. The site‐specific disulfide introduction into the flexible N‐terminal loop of natural Kazal‐type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.