Document Detail


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.
MedLine Citation:
PMID:  21644249     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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.
Authors:
Hikaru Hemmi; Takashi Kumazaki; Shuichi Kojima; Takuya Yoshida; Tadayasu Ohkubo; Hideyoshi Yokosawa; Kin-Ichiro Miura; Yuji Kobayashi
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-6-6
Journal Detail:
Title:  Journal of peptide science : an official publication of the European Peptide Society     Volume:  -     ISSN:  1099-1387     ISO Abbreviation:  -     Publication Date:  2011 Jun 
Date Detail:
Created Date:  2011-6-6     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9506309     Medline TA:  J Pept Sci     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.
Affiliation:
National Food Research Institute, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
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